Microbial production of steviol glycosides

ABSTRACT

The invention provides methods for making steviol glycosides, including RebM and glycosylation products that are minor products in stevia leaves, and provides enzymes, encoding polynucleotides, and host cells for use in these methods. The invention provides engineered enzymes and engineered host cells for producing steviol glycosylation products, such as RebM, at high purity and/or yield. The invention further provides methods of making products containing steviol glycosides, such as RebM, including food products, beverages, oral care products, sweeteners, and flavoring products.

RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 62/075,644, filed Nov. 5, 2014, which is herebyincorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to enzymes, including engineered enzymes,encoding polynucleotides, host cells, and methods for producing steviolglycosides.

BACKGROUND

High intensity sweeteners possess a sweetness level that is many timesgreater than the sweetness level of sucrose. They are essentiallynon-caloric and are commonly used in diet and reduced-calorie products,including foods and beverages. High intensity sweeteners do not elicit aglycemic response, making them suitable for use in products targeted todiabetics and others interested in controlling their intake ofcarbohydrates.

Steviol glycosides are a class of compounds found in the leaves ofStevia rebaudiana Bertoni, a perennial shrub of the Asteraceae(Compositae) family native to certain regions of South America. They arecharacterized structurally by a single base, steviol, differing by thepresence of carbohydrate residues at positions C13 and C19. Theyaccumulate in Stevia leaves, composing approximately 10% to 20% of thetotal dry weight. On a dry weight basis, the four major glycosides foundin the leaves of Stevia typically include stevioside (9.1%),rebaudioside A (3.8%), rebaudioside C (0.6-1.0%) and dulcoside A (0.3%).Other known steviol glycosides include rebaudioside B, C, D, E, F and M,steviolbioside and rubusoside.

The minor glycosylation product rebaudioside M is estimated to be about200-350 times more potent than sucrose, and is described as possessing aclean, sweet taste with a slightly bitter or licorice aftertaste.Prakash I. et al., Development of Next Generation Stevia Sweetener:Rebaudioside M, Foods 3(1), 162-175 (2014). RebM is of great interest tothe global food industry.

Although methods are known for preparing steviol glycosides from Steviarebaudiana, many of these methods are unsuitable for use commerciallyand/or are not sustainable. Accordingly, there remains a need forsimple, efficient, and economical methods for preparing compositionscomprising steviol glycosides, including highly purified steviolglycoside compositions. Further, methods are needed for producingsubstantial amounts of the minor glycosylation products, includingproducts having a plurality of glycosylations, such as Reb A, Reb D, RebE, Reb I, RebM, and others.

SUMMARY OF THE INVENTION

In various aspects, the invention provides methods for making steviolglycosides, including Reb D and RebM and glycosylation products that areminor products in stevia leaves, and provides enzymes, encodingpolynucleotides, and host cells for use in these methods. The inventionprovides engineered enzymes and engineered host cells for producingsteviol glycosylation products, such as Reb D and RebM, at high purityand/or yield. The invention further provides methods of making productscontaining steviol glycosides, where the products include food products,beverages, oral care products, sweeteners, flavoring products, amongothers.

In various aspects and embodiments, the invention provides enzymes,encoding polynucleotides, host cells, and methods for producing steviolglycosides having a plurality of glycosylations at C13 and/or C19. Thesteviol glycosides may have 2, 3, 4, 5, 6, 7, 8 or more glycosylations.In various embodiments, the glycosylations are selected from: C13-O,C19-O, 1-2′ (at C-13 and/or C19 of steviol), and 1-3′ (at C13 and/or C19of steviol). Exemplary enzymes to perform these glycosylations arelisted in Table 8, and include enzymes that catalyze C13-Oglycosylations of steviol (e.g., SrUGT85C2), C19-O glycosylations ofsteviol (e.g., SrUGT74G1), 1-2′ glycosylations of steviol glycosides(e.g., SrUGT91D1, SrUGT91D2, OsUGT1-2), and 1-3′ glycosylations ofsteviol glycosides (e.g., SrUGT76G1). Numerous derivatives that can beused in various embodiments are disclosed herein, including enzymesidentified herein as MbUGTc13 (SEQ ID NO:51), MbUGTc19 (SEQ ID NO:8),MbUGTc19-2 (SEQ ID NO:46), MbUGT1-2 (SEQ ID NO:9), MbUGT1,2-2 (SEQ IDNO:45), and MbUGT1-3 (SEQ ID NO:10), and derivatives thereof. In someembodiments, the invention provides host cells that express at least 2,3, or 4 UGT enzymes for performing these glycosylations in vivo on thesteviol substrate. Various steviol glycoside products that can beproduced according to embodiments of the invention are shown in FIGS.28-31 and Table 10, and these include Reb M, Reb D, Reb E, and Reb I. Inaccordance with embodiments of the invention, these steviol glycosidescan be produced at high yields in bacterial host cells, such as E. coli,including at temperatures suitable for E. coli growth and metabolism.

In some aspects, the invention provides modified UGT enzymes having anincrease in 1-2′ glycosylating activity at C19 of Rebaudioside A (RebA)as compared to its parent UGT enzyme, and without substantial loss of1-2′ glycosylating activity at C13 of steviolmonoside. Such enzymes canprovide for increased carbon flux to RebD. Further, the inventionprovides modified UGT enzymes having an increase in 1-3′ glycosylatingactivity at C19 of Rebaudioside D (RebD) as compared to its parent UGTenzyme, without substantial loss of 1-3′ glycosylating activity at C13of stevioside. Such enzymes can provide for increased carbon flux toRebM.

In accordance with the present disclosure, production of steviolglycosides is engineered in host cells through the production of variouspathway modules from glycolysis to steviol, and further to steviolglycosides, and which can be optimized and balanced to promote carbonflux to steviol and then to Reb D or RebM (or other glycosylationproduct) as the main glycosylation product.

In another aspect, the invention provides a method for making RebD. Themethod comprises providing a host cell producing RebD from steviolthrough a plurality of uridine diphosphate dependent glycosyltransferaseenzymes (UGT), and culturing the host cell under conditions forproducing the RebD. The UGT enzymes comprise a modified UGT enzymehaving an increase in 1-2′ glycosylating activity at C19 of RebaudiosideA (RebA) as compared to its parent UGT enzyme, without substantial lossof 1-2′ glycosylating activity at C13 of steviolmonoside. In certainembodiments, the 1-2′ glycosylating activity at C19 of Rebaudioside A(RebA) is equal to or better than the 1-2′ glycosylating activity at C13of steviolmonoside.

In another aspect, the invention provides a method for making RebM. Themethod comprises providing a host cell producing RebM from steviolthrough a plurality of uridine diphosphate dependent glycosyltransferaseenzymes (UGT), and culturing the host cell under conditions forproducing the RebM. The UGT enzymes comprise one or more of: (a) amodified UGT enzyme having an increase in 1-2′ glycosylating activity atC19 of Rebaudioside A (RebA) as compared to its parent UGT enzyme,without substantial loss of 1-2′ glycosylating activity at C13 ofsteviolmonoside; and (b) a modified UGT enzyme having an increase in1-3′ glycosylating activity at C19 of Rebaudioside D (RebD) as comparedto its parent UGT enzyme, without substantial loss of 1-3′ glycosylatingactivity at C13 of stevioside. In certain embodiments, the 1-2′glycosylating activity at C19 of Rebaudioside A (RebA) is equal to orbetter than the 1-2′ glycosylating activity at C13 of steviolmonoside.Alternatively or in addition, the 1-3′ glycosylating activity at C19 ofRebaudioside D (RebD) is equal to or better than the 1-3′ glycosylatingactivity at C13 of stevioside.

In some embodiments, the invention provides modified SrUGT76G1 enzymes,which provide for 1-3′ glycosylating activity of stevioside and RebD,including enzymes having an amino acid substitution at position 200 ofthe wild type enzyme (e.g., L200A or L200G), which exhibit substantialimprovement in activity.

In other aspects and embodiments, the invention provides circularpermutants of UGT enzymes (as well as encoding polynucleotides), whichcan provide novel substrate specificities, product profiles, andreaction kinetics over the wild-type enzymes. The circular permutantscan be expressed in host cells for production of steviol glycosides asdescribed herein. Thus, in various embodiments the microbial cellexpresses at least one UGT enzyme that is a circular permutant of awild-type or parent UGT enzyme. A circular permutant retains the samebasic fold of the parent enzyme, but has a different position of theN-terminus (e.g., “cut-site”), with the original N- and C-terminiconnected, optionally by a linking sequence. For example, in thecircular permutants, the N-terminal Methionine is positioned at a sitein the protein other than the natural N-terminus. For example, theinvention provides circular permutants of OsUGT1-2 and SrUGT74G1, whichcan be further modified as described herein for production ofglycosylation products of steviol.

In another aspect, the invention provides a method for production ofsteviol glycosides having at least 4 glycosylations in E. coli. Inaccordance with the invention, the E. coli cell comprises a plurality ofUGT enzymes, which may include one or more enzymes described herein,that together perform at least 4, at least 5, or at least 6, sequentialglycosylation reactions. As disclosed herein, the glycosylationsubstrates and lower glycosylation products accumulate in the E. colicell sufficiently to allow downstream reactions to proceed at anacceptable rate, with a high majority of the glycosylation productsultimately accumulating extracellularly. The steviol glycosides can bepurified from media components. Thus, E. coli is a desirable host forproduction of steviol glycosides that require several glycosylationreactions of the steviol scaffold.

In still other aspects, the invention provides methods for production ofsteviol glycosides (including RebM, Reb D, Reb E, Reb I, and others) inE. coli. While many of the enzymes known for production of steviol inhost cells are plant enzymes, which often have optimal temperatures inthe range of 20-24° C., E. coli growth rate and metabolism are optimalat higher temperatures. The present disclosure enables production ofsteviol glycosides at high yield in E. coli, by enabling enzymeproductivity at temperatures above 24° C., such as from 24° C. to 37°C., or from 27° C. to 37° C., or from 30° C. to 37° C. In variousembodiments, the disclosure provides alternative or engineered GGPPS,KS, CPPS, KO, and KAH enzymes for production of steviol or steviolglycosides in E. coli or other microbial host.

Other aspects and embodiments of the invention will be apparent from thefollowing detailed disclosure.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemical structure of Rebaudioside M (RebM), a minorcomponent of the steviol glycoside family, and which is a derivative ofthe diterpenoid steviol (box) with six glucosyl-modification groups.

FIG. 2 shows pathway modules to RebM. Glycolysis and MEP pathways aretreated as one module, and the downstream kaurene biosynthesis pathwayis shown as the second module. Biosynthesis of steviol is shown as thethird module. The fourth module is for the glycosylation of steviol andthe RebM biosynthetic pathway. The fifth module to support enhancedUDP-glucose production is also shown.

FIG. 3 shows an exemplary pathway for steviol glycoside production,including to RebM. PsCKS is a bifunctional copalyl diphosphate andkaurene synthase (from Phaeosphaeria sp.) which acts on geranylgeranyldiphosphate (synthesized from IPP and DMAPP by Taxus canadensis GGPPsynthase, not shown). SrKO is Stevia rebaudiana kaurene oxidase andAtKAH is an Arabidopsis thaliana P450 with steviol monooxygenaseactivity. Solid arrows are known UGT activities. Arrows with dotted lineborders are predicted reactions based on demonstrated activities onother substrates in vitro. MbUGT1-2 is a novel UGT enzyme designed inthis disclosure.

FIG. 4 shows kaurene production profiles from engineered E. coli cells.(A) and (B) are kaurene production from CPPS/KS enzymes selected fromplant Stevia rebaudiana (SrCPPS and SrKS) and Physcomitrella patens(PpCK), respectively. (C) and (D) are strains constructed with enzymesselected from fungus species Gibberella fujikuroi (GfCK) andPhaeosphaeria sp. (PsCK), respectively.

FIG. 5 shows GC profiles from strains constructed from different KSenzymes. The pathway is shown in FIG. 2. The figure in the box (leftinset) is the magnified chromatograph to show the byproductaccumulation. The GC profile and corresponding MS spectra show that theKS enzymes can be non-specific vis-à-vis product profile. Otherterpenoid byproducts were produced with similar MS characteristics askaurene. In all three pathways the major product is kaurene. Theauthenticity of kaurene is confirmed by comparison to MS spectra and NMRdata reported in previously published literature. The MS spectra fromall the byproduct show a characteristic 272 molecular ion.

FIG. 6 shows the product profile from engineered strains. Shown are theproduction profiles from different downstream pathway expression levelsunder different upstream pathway modulation. The byproducts are the sameas those shown in FIG. 5. Genotype details of each strain are in Table2.

FIG. 7 shows that a strain (Strain 47 in table 2, Ch1TrcMEP-Ch1T7PsCKG)with properly balanced modules enabling kaurene biosynthesis, is capableof multi-gram-per-liter scale productivity of kaurene in a 2 Lbioreactor.

FIG. 8 shows that indole accumulation is inversely correlated to kaureneproduction across engineered strains.

FIG. 9 shows redesign and characterization of SrKO enzyme. (A) TheN-terminal transmembrane region analysis and truncations withmodifications to SrKO. Alignments include SrKO (SEQ ID NO:53),17a-t4srKO (SEQ ID NO:54), 17a-t20srKO (SEQ ID NO:55), 17a-t36srKO (SEQID NO:56), and MA-t39srKO (SEQ ID NO:57). MALLLAVF (SEQ ID NO: 47) tagis also shown. (B) Schematics of designed SrKO/SrCPR enzyme constructs.(C) Protein expression from different engineered constructs in E. coli:(1) WT, (2) WT+(MA)KO-O-CPR, (3) WT+(MA)KO-O-CPR, (4) WT+(MA)KO-L-CPR,(5) WT+(MA)KO-L-CPR, (6) WT+(8RP)KO-O-CPR, (7) WT+(8RP)KO-O-CPR, (8)WT+(8RP)KO-L-CPR, (9) WT+(8RP)KO-L-CPR.

FIG. 10 shows the kaurenoic acid productivity of SrKO in linker oroperon configuration with SrCPR in strain 47 background.

FIG. 11 illustrates the MMME landscape exploration of SrKO constructsunder varying plasmid copy numbers and promoter strength. Imbalancedmodules show less or no kaurenoic acid accumulation, with an associatedincrease in upstream kaurene accumulation instead.

FIG. 12 shows design of a CYP450 expression module to screen for optimumenzyme variants, N-terminal truncations, and point mutations of KO, KAH,or CPR genes. The two P450s and the CPR enzyme are expressed in apolycistronic operon under various promoter strengths in either plasmidor chromosomally-integrated format.

FIG. 13 shows point mutants of AtKAH enzyme, as represented byfold-change in kaurenoic acid hydroxylase activity relative to wild-typeAtKAH.

FIG. 14 shows a series of engineered AtKAH that demonstrate improvedsteviol productivity and eventual complete conversion of kaurenoic acidto steviol.

FIG. 15 shows that, in a properly balanced module, the two P450s (AtKAHand SrKO) and the co-factor CYP450 reductase (SrCPR) are capable ofcomplete conversion of kaurene through kaurenoic acid through tosteviol.

FIG. 16 demonstrates increased UDP-glucose production in E. coli using amodel system: glycosylation of a small molecule caffeic acid withterpene producing E. coli strains engineered for increased UDP-glucoseproduction, producing caffeic acid 3-glucoside using Vitis viniferaglycosyltransferase 2 (VvGT2) overexpressed from a pET plasmid. Theimprovement in glycosylated caffeic acid titers compared to theunmodified background strain shows an increase in the UDP-glucosesubstrate pool to support glycosylation. Strain 1 is Strain 47 (Table 2)with knock-outs of the galactose catabolic module (galETKM), UDP-sugarpyrophosphatase (ushA), phosphoglucomutase (pgm), glycose-1 phosphatase(agp), β-galactosidase (lacZ), and overexpressing sucrose phosphorylase(spl) under the Trc promoter (see Table 7). Strain 2 is Strain 47 (Table2) with knock-outs of the galactose catabolic module (galETKM),UDP-sugar pyrophosphatase (ushA), phosphoglucomutase (pgm), glycose-1phosphatase (agp), β-galactosidase (lacZ), and overexpressing andsucrose phosphorylase (spl) under the T7 promoter (see Table 7).

FIG. 17 shows the process for identification of an optimum glycosylationmodule incorporating all four UGT activities. All 24 possiblecombinations are rapidly assembled in three different plasmids, enablingexpression at three different levels, for a total of 72 potentialconstructs.

FIG. 18 shows in vivo production of RebM. (A) Product titers of steviolglycoside from E. coli culture. (B) LC/MS trace showing RebMidentification. Negative control strain has been modified to producesteviol and increased UDP-glucose pools, while 4UGT strain is thenegative control strain plus four UGTs.

FIG. 19 shows a homology model of OsUGT1-2 (1-2′ glycosylating enzymefrom rice, Oryza sativa), as a starting point for circular permutantdesign.

FIG. 20 shows linkers for UGT circular permutants, to connect thenatural N and C-termini. Three different linkers are shown: (A)YKDDSGYSSSYAAAAGM (SEQ ID NO:48) attaching the existing sequence, (B)YKDAAGM (SEQ ID NO:49), creating an intermediate-length loop, and (C)YGSGM (SEQ ID NO:50), creating a minimal loop.

FIG. 21 illustrates criteria for selection of new N- and C-termini forthe UGT circular permutant. Positions for new termini should be: (1)solvent exposed and away from the active site to minimize perturbation,(2) close to the middle of the sequence to maximize difference with theparental sequence, and (3) have amino acids often found at existingcircular permutant division points (Lo, et al., 2012, PLoS One7(2):e31791). New N-termini at G198, K240, G250, and G259 fit thesecriteria.

FIG. 22 shows 1-2′ glycosylating activity for the first round ofcircular permutants of OsUGT1-2. The numbers indicate the location ofthe cut site in the parental sequence used to generate novel positionsfor N- and C-termini, while the L/M/S designation describes thelong/medium/short linkers (which are described in FIG. 20).

FIG. 23 shows refinement of the 1-2′ UGT circular permutant (MbUGT1-2).Modifications to the cut site and linker length demonstratesignificantly enhanced activity on at least one of the substratespossible for this enzyme. Number before L (eg. xxxL) indicates new cutsite position, while number after L (eg. 1Lxx) indicates a new linkerlength in background with the 198 cut site [BL21=negative no UGTcontrol].

FIG. 24 shows point mutations in the MbUGT1-2 enzyme. Point mutationsshow increased activity on substrate steviolmonoside, demonstrating thepotential for improving UGT enzymes generated by circular permutization[BL21=negative no UGT control].

FIG. 25 shows point mutations in the MbUGT1-2 enzyme. Point mutationsshow increased activity on substrate rebaudioside A, demonstrating thepotential for improving UGT enzymes generated by circular permutization.[BL21=negative no UGT control].

FIG. 26 shows that point mutations that are beneficial to the MbUGT1-2enzyme do not, when translated to the appropriate amino acid residue inthe parental UGT enzyme, result in neutral or even deleterious effectson activity. This demonstrates that circular permutants have thepotential for unique improvements and evolution compared to the parentenzyme, brought about by shuffling of the sequence into a novelarrangement not previously selected for by natural selection.[BL21=negative no UGT control].

FIG. 27 shows a chimeric UGT with C13-O-glycosylating activity, createdby fusing the N-terminus of SrUGT85C2 and the C-terminus of SrUGT76G1.

FIG. 28 is a summary of possible reactions (marked by arrows) catalyzedby SrUGT85C2 (i.e., C13-O-glycosylations).

FIG. 29 is a summary of possible reactions (marked by arrows) catalyzedby SrUGT74G1 (i.e., C19-O-glycosylations).

FIG. 30 is a summary of possible reactions (marked by arrows) catalyzedby MbUGT1-2 (i.e., 1-2-glycosylations).

FIG. 31 is a summary of possible reactions (marked by arrows) catalyzedby SrUGT76G1 (i.e., 1-3-glycosylations).

FIG. 32 shows point mutations in SrUGT85C2 enzyme versus alteredactivity on steviol substrate. [BL21=negative no UGT control].

FIG. 33 shows point mutations in SrUGT85C2 enzyme versus alteredactivity on C19-glucopyranosyl steviol substrate. [BL21=negative no UGTcontrol].

FIG. 34 shows C19-O-glycosylating activity for the first round ofcircular permutants of SrUGT74G1. The numbers indicate the location ofthe cut site in the parental sequence used to generate novel N- andC-termini, while the wt/L/S designation describes wild-type/long/shortlinkers (where ‘wt’ indicates a simple fusion of existing N- andC-termini sequences with no alteration).

FIG. 35 shows point mutations in SrUGT76G1 enzyme versus alteredactivity on stevioside substrate. [BL21=negative no UGT control].

FIG. 36 shows point mutations in SrUGT76G1 enzyme versus alteredactivity on rebaudioside D substrate. [BL21=negative no UGT control].

FIG. 37 shows 1-3′ glycosylating activity for the first round ofcircular permutants of SrUGT76G1. The numbers indicate the location ofthe cut site in the parental sequence used to generate novel N- andC-termini, while the L/S designation describes long/short linkers.

FIG. 38 shows UGT alignment and secondary structure, anchored to 2VCEwhich is an Arabidopsis UGT with a solved crystal structure. Alignmentsinclude 2VCE (SEQ ID NO:58), 2ACW (SEQ ID NO:59), 2C1Z (SEQ ID NO:60),2PQ6 (SEQ ID NO:61), 3HBF (SEQ ID NO:62), 3WC4 (SEQ ID NO:63), SrUGT85C2(SEQ ID NO:1), SrUGT74G1 (SEQ ID NO:2), Q0DPB7-ORYSJ is OsUGT1-2 (SEQ IDNO:7), SrUGT76G1 (SEQ ID NO:3). Boxed is the position of the 76G1-L200Apoint mutation, which promotes significantly improved activity. Alsoshown in boxes is the conserved PSPG motif

FIG. 39 shows alternate GGPPS enzymes tested in vivo for performance at22° C., 30° C., and 37° C.

FIG. 40 shows alternate CPPS/KS pairs tested in vivo for performance at22° C., 30° C., and 37° C.

FIG. 41 shows the titer of Reb M in comparison to steviol and otherglycosylation products, using a selected strain at 22° C.

FIG. 42 shows that the majority of Reb M accumulates extracellularly.Left Panel shows the titer of Reb M and steviol glycosides inside andoutside of the cell. Right Panel shows the same data as the percent ofeach compound observed extracellularly.

FIG. 43 shows screening of UGT85C2 mutants at 22, 30, and 34° C., basedon production of steviolmonoside. Panels A and B show the panel ofmutants at 34° C., and Panel C shows select mutations screened forsteviolmonoside production at 22, 30, and 34° C.

FIG. 44 shows screening of 74G1 circular permutants for activity at 30and 34° C. Panel 44A shows activity on Steviol, and Panel 44B showsactivity on steviolbioside.

FIG. 45 shows screening of AtKAH point mutants for activity at 22, 26,and 30° C.

FIG. 46 shows in vitro screening of MbUGT1-2 recombination mutants at 30and 34° C. Panel A shows conversion of Reb A to Reb D. Panel B showsconversion of Steviolmonoside to 13C Steviolbioside.

FIG. 47 shows kaurene production at 30° C. across various moduleconstructs.

FIG. 48 shows kaurene production at 34° C. across various moduleconstructs.

FIG. 49 shows production of Steviol at 30° C. across a library of AtKAHpoint mutations.

FIG. 50 shows production of Steviol at 34° C. across a library of AtKAHpoint mutations.

FIG. 51 shows activities of MbUGT1-2 circular permutants at 30° C., 34°C., and 37° C. Panel (A) shows conversion of Reb A to Reb D. Panel (B)shows conversion of Steviolmonoside to 13C Steviolbioside.

FIG. 52 shows activities of UGT85C2 mutants for conversion of Steviol to13C Steviolmonoside at 30° C., 34° C., and 37° C.

FIG. 53 shows activities of UGT76G1 mutants for conversion of Reb D to13C Reb M at 30° C., 34° C., and 37° C.

FIG. 54 shows activities of UGT74G1 circular permutants for conversionof Steviolbioside to 13C Stevioside at 30° C., 34° C., and 37° C.

DETAILED DESCRIPTION OF THE INVENTION

In various aspects, the invention provides methods for making steviolglycosides, including RebM, RebD, and glycosylation products that areminor products in stevia leaves, and provides enzymes, encodingpolynucleotides, and host cells for use in these methods. The inventionprovides engineered enzymes and engineered host cells for producingsteviol glycosylation products at high purity and/or yield. Theinvention further provides methods of making products containing steviolglycosides, such as RebM or RebD, including food products, beverages,oral care products, sweeteners, flavoring products, among others. Suchsteviol glycoside-containing products can be made at reduced cost byvirtue of this disclosure.

RebM is illustrated in FIG. 1, with the steviol scaffold (a diterpenoid)shown boxed. RebM contains six glycosylations: (1) a C13O-glycosylation, (2) a C13 1-2′ glycosylation, (3) a C13 1-3′glycosylation, (4) a C19 O-glycosylation, (5) a C19 1-2′ glycosylation,and (6) a C19 1-3′ glycosylation. Pathways from geranylgeranylpyrophosphate (GGPP) to RebM are illustrated in FIG. 3. GGPP producedfrom IPP and DMAPP (products of the MEP or MVA pathways), is convertedto kaurene by the action of copalyl synthase and kaurene synthase, whichcan be present as a bifunctional enzyme in some embodiments. Steviol isproduced from kaurene by the action of two P450 enzymes, kaurene oxidaseand kaurenoic acid hydroxylase, which are regenerated by one or moreP450 reductase enzymes. After production of steviol, a series ofglycosylation reactions at C13 and C19 are capable of producing varioussteviol glycoside products, including the hexaglycosylated RebM. Variousother glycosylation products are possible (as shown in FIG. 3), and asillustrated in FIGS. 28-31, known UGT glycosylation enzymes are eachcapable of acting on a number of substrates. Thus the fidelity, relativereaction rates, expression levels, and availability of substrate willaffect the relative yields of the glycosylation products. For example,both UGT91D2 and OsUGT1-2 are 1-2′ glycosylating enzymes that canproduce steviolbioside from steviolmonoside (by action at C13), as wellas RebD from RebA (by action at C19). Further, UGT76G1 is a 1-3′glycosylating enzyme that can produce RebA from stevioside (by action atC13), as well as RebM from RebD (by action at C19). Tables 8, 9, and 10show the various possible steviol glycosides that may result from thesix glycosylation reactions, as well as enzymes for each reaction. Table1 lists various enzymes that may be used for the production of steviolglycosides. Amino acid sequences are also provided herewith, each ofwhich can optionally include an alanine inserted or substituted atposition 2 to decrease turnover in the cell. Certain GGPPS sequencesfurther contain two additional residues (VD) at the end of the sequence,which are not believed to have any deleterious effect, and may beomitted in certain embodiments.

Thus, in some aspects, the invention provides enzymes, encodingpolynucleotides, and host cells engineered for maximizing the productionof the desired steviol glycoside (e.g., RebM). For example, thisdisclosure provides modified UGT enzymes having an increase in 1-2′glycosylating activity at C19 of Rebaudioside A (RebA) as compared tothe parent UGT enzyme, and without substantial loss of 1-2′glycosylating activity at C13 of steviolmonoside. Such enzymes mayprovide for increased carbon flux to RebD. Further, this disclosureprovides modified UGT enzymes having an increase in 1-3′ glycosylatingactivity at C19 of Rebaudioside D (RebD) as compared to the parent UGTenzyme, without substantial loss of 1-3′ glycosylating activity at C13of stevioside. Such enzymes may provide for increased carbon flux toRebM. In some aspects and embodiments, and without wishing to be boundby theory, the invention provides for modified UGT enzymes withsubstrate binding pockets that are better able to accommodate substrates(including larger substrates), thereby increasing the rate of activity(e.g., rate of substrate binding and turnover) with more highlyglycosylated steviol substrates such as RebA or RebD.

The invention in some aspects provides for a controlled glycosylationpathway that produces largely RebM as a glycosylation product. Forexample, in some embodiments, the invention provides a method for makingRebM in microbial cells, where the RebM:RebD ratio is greater than about1:1, or greater than about 1:0.5, or greater than about 1:0.25, orgreater than about 10:1, or greater than about 25:1, or greater thanabout 50:1. In some embodiments, RebD is produced at less than about20%, or at less than about 10%, or at less than about 5%, or at lessthan about 1% of the RebM yield, or is not detectable in the isolatedsteviol glycosylation products. Because RebD can be difficult toseparate from RebM, or can add significant purification costs if suchseparation is necessary, products with low levels of RebD are desirablein some embodiments. In some embodiments, RebM represents at least about25% by weight of the steviol glycosylation products produced by thecell, or at least about 50% by weight of the glycosylation products, orat least about 75% by weight of the glycosylation products, or at leastabout 80% by weight of the glycosylation products, or at least about 85%by weight of the glycosylation products, or at least about 90% by weightof the glycosylation products, or at least about 95% by weight of thesteviol glycosylation products.

The glycosylation pathways involve a 13-O glycosylation, a 19-Oglycosylation, as well as one or more 1-2′ glycosylations and/or one ormore 1-3′ glycosylations at C13 and/or C19 of steviol. The term “steviolglycoside(s)” refers to a glycoside of steviol, including, but notlimited to, steviolmonoside, steviolbioside, rubusoside, dulcoside B,dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C,rebaudioside F, rebaudioside A, rebaudioside E, rebaudioside H,rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M,rebaudioside D, rebaudioside N, rebaudioside O. The chemical identitiesof these steviol glycosides are known, and are described for example, inTable 10, as well as in WO 2014/122227, which is hereby incorporated byreference in its entirety.

In accordance with the present disclosure, production of steviolglycosides is engineered in host cells through the production of variouspathway “modules,” as illustrated in FIG. 2, and which can be optimizedand balanced to promote carbon flux to steviol and then a desiredglycosylation product (such as RebM or RebD) as the main glycosylationproduct. By grouping enzymes with similar turnovers into a subset, ormodule, and equalizing the turnover of the different subsets byadjusting concentrations/activities of enzymes, the ratio of pathwayturnover to resource expenditure can be optimized.

The first pathway module comprises enzymes in the MEP or MVA pathways,which produce IPP and DMAPP. The MEP and MVA pathways may be endogenousto the organism, and these pathways may be increased and balanced withdownstream pathways by providing duplicate copies of certainrate-limiting enzymes. IPP and DMAPP act as a substrate for theproduction of (−)-kaurene (e.g., by separate copalyl synthase andkaurene synthase enzymes, or a bifunctional enzyme), which is the secondpathway module. A third pathway module converts (−)-kaurenoic acid tosteviol by the action of two P450 enzymes (e.g., kaurene oxidase (KO)and kaurenoic acid hydroxylase (KAH)) and one or more P450 reductaseenzymes. Exemplary enzymes that catalyze production of GGPP and itsconversion through to steviol are listed in Table 1. Steviol is thenglycosylated to the final product by a UDP enzyme module. An additionalmodule includes genes that enhance production of the UDP-glucosesubstrate. In various embodiments of the invention, these modules areeach present as mono- or poly-cistronic operons, which are each harboredon plasmids or are chromosomally integrated. In certain embodiments, themodules are configured for increased production of the desiredend-product.

In one aspect, the invention provides a method for making a steviolglycoside, which is optionally RebM or RebD. The method comprisesproviding a host cell producing the steviol glycoside from steviolthrough a plurality of uridine diphosphate dependent glycosyltransferaseenzymes (UGT), and culturing the host cell under conditions forproducing the steviol glycoside. The UGT enzymes comprise one or moreof: (a) a modified UGT enzyme having an increase in 1-2′ glycosylatingactivity at C19 of Rebaudioside A (RebA) as compared to its parent UGTenzyme, without substantial loss of 1-2′ glycosylating activity at C13of steviolmonoside (e.g., when evaluated at 22° C., 27° C., or 30° C.);and (b) a modified UGT enzyme having an increase in 1-3′ glycosylatingactivity at C19 of Rebaudioside D (RebD) as compared to its parent UGTenzyme, without substantial loss of 1-3′ glycosylating activity at C13of stevioside (e.g., when evaluated at 22° C., 27° C., or 30° C.).

In certain embodiments, the 1-2′ glycosylating activity at C19 ofRebaudioside A (RebA) is equal to or better than the 1-2′ glycosylatingactivity at C13 of steviolmonoside. Alternatively or in addition, the1-3′ glycosylating activity at C19 of Rebaudioside D (RebD) is equal toor better than the 1-3′ glycosylating activity at C13 of stevioside.

In some embodiments, the modified UGT enzyme having 1-2′ glycosylatingactivity and/or the UGT enzyme having 1-3′ glycosylating activity doesnot exhibit a substantial loss of activity at C13, as compared to theparent enzyme. For example, the modified enzyme retains at least 50% ofits activity at C13, or at least about 75% of its activity at C13, or atleast about 80%, at least about 90%, or at least about 95% of itsactivity at C13 as compared to the parent (e.g., wild-type) enzyme(e.g., when evaluated at 22° C., 27° C., or 30° C.). In someembodiments, the enzyme has improved activity at C13, such as at least2-fold or at least 3-fold improved activity at C13. The loss of, orimprovement in, a glycosylation activity can be determined in vitro, forexample in cell extracts with the substrate of interest added, or otherin vitro or in vivo assay. For example, relative reaction rates may bedetermined in a strain that produces the steviol or steviol glycosidesubstrate(s) of interest. Exemplary assays for quantifying glycosylationactivity are disclosed herein as well as in WO 2014/122227, which ishereby incorporated by reference.

While in some embodiments, the 1-2′ and 1-3′ glycosylation activities atC13 are sufficiently functional with the enzyme that performs thesereactions at C19 (e.g., without any additional enzyme to perform theseenzymatic steps), in other embodiments, the cell further expresses anenzyme to perform 1-2′ and/or 1-3′ glycosylation at C13. In someembodiments, a second enzyme is engineered to perform the 1-2′ and/or1-3′ reactions at C13, even with loss of activity at C19.

In some embodiments, the cell expresses only one UGT enzyme having 1-2′glycosylating activity at C13 of steviolmonoside, and/or expresses onlyone UGT enzyme having 1-3′ glycosylating activity at C13 of stevioside.In such embodiments, the enzyme can be engineered to enhance thereaction at C19, thereby pulling product toward C19 glycosylationproducts such as RebM, without the need for expression of additionalenzymes that place a further metabolic burden on the cell.

In aspects and embodiments, the invention provides circular permutantsof UGT enzymes (as well as encoding polynucleotides and methods ofmaking circular permutants of UGT enzymes), which can provide novelsubstrate specificities, product profiles, and reaction kinetics overthe parent (e.g., wild-type) enzymes. Without wishing to be bound bytheory, circular permutants provide the opportunity to make the UGTbinding pocket more open or accessible for larger substrates, such assteviol substrates having one or more glycosyl groups. In this manner,the invention allows for the glycosylation reactions on moreglycosylated forms of steviol to proceed at rates similar to or evengreater than reactions on less glycosylated (and thus smaller)substrates. The circular permutants can be expressed in host cells forproduction of steviol glycosides as described herein. Thus, in variousembodiments the microbial cell producing the steviol glycoside (e.g.,RebM or RebD) expresses at least one UGT enzyme that is a circularpermutant of a parent (e.g., wild-type) UGT enzyme.

A circular permutant retains the same basic fold of the parent enzyme,but has a different position of the natural N-terminus (e.g.,“cut-site”), with the original N- and C-termini connected, optionally bya linking sequence. An exemplary structure of a UGT enzyme (e.g., basedon OsUGT1-2) is shown in FIG. 20. A UGT alignment and secondarystructure elements are shown in FIG. 38. For each circular permutant,the cut-site can be described with reference to the correspondingposition of the parent sequence (e.g., wild-type sequence), by alignmentof the permutant's N-terminal amino acids (e.g., N-terminal 50 or 100amino acids) with the parent or wild-type sequence. As used herein, the“cut site” of a given circular permutant refers to the original positionof the amino acid that is positioned at position 2 in the circularpermutant (e.g., after the initiating Met), or position 3 of thecircular permutant when an Alanine is inserted at position 2 to decreaseprotein turnover. Alignments for comparing global UGT sequences shouldbe anchored around the conserved PSPG motif shown in FIG. 38. The PSPG(plant secondary product glycosyltransferase) motif is a conservedregion within plant UGTs that plays a role in binding thenucleotide-diphosphate-sugar donor molecule. Gachon et al., Plantsecondary metabolism glycosyltransferases: the emerging functionalanalysis, Trends Plant Sci. 10:542-549 (2005). The most conservedresidues in this motif in the UDPGT family show the pattern:WXPQXXXLXHXXXXAFXXHXGXXXXXEXXXXGXPXXXXPXFXXQ (SEQ ID NO:52), of whichthe underlined histidine makes a critical contact to the diphosphateregion. Finn R D, et al. Pfam: the protein families database, NucleicAcids Res. 42:D222-230 (2014). Further, alignment around this motif isuseful for describing point mutations that translate to beneficialproperties for the UGT proteins as a class. For example, anchoringalignments to the tryptophan at the beginning of the motif, or theimportant histidine in the middle, may be used to describe pointmutations relative to this sequence, which will be universal in plantGT1 UDP-glucose glycosyltransferases.

In some embodiments, the circular permutant is a circular permutant ofUGT85C2 from Stevia rebaudiana. SrUGT85C2 is provided herein as SEQ IDNO:1. In some embodiments, the circular permutant is a circularpermutant of OsUGT1-2 (SEQ ID NO:7). In some embodiments, the circularpermutant is of UGT91D2 of Stevia rebaudiana (SEQ ID NO:5). In someembodiments, the circular permutant is of UGT74G1 of Stevia rebaudiana(SEQ ID NO:2). In some embodiments, the circular permutant is of UGT76G1of Stevia rebaudiana (SEQ ID NO:3). In this manner, by changing theposition of the N-terminus of the UGT enzyme, enzymes with novelsubstrate specificities and activity profiles can be created. Forexample, in some embodiments, the cut site is between amino acids 150 to300, or in some embodiments between amino acids 190 and 260, or in someembodiments between residues 190 and 210, when the N-terminus of thecircular permutant (e.g., N-terminal 50 amino acids) is aligned with theparent or wild-type enzyme. In other embodiments, the circular permutanthas a cut site between amino acids 245 and 280 (e.g., position 272), orbetween amino acids 260 to 275, when the N-terminal 50 amino acids ofthe circular permutant are aligned with the parent or wild-type enzyme.In some embodiments, the new N-terminus is placed between localsecondary structure elements (such as α-helices or β-sheets), and/or isplaced at a loop structure of the wild-type enzyme. When selecting thedesired position of the N-terminus, a Met is added to the cut-site asthe initiating amino acid, and an Ala is optionally placed at the secondposition to decrease cellular turnover. The natural N and C-termini arelinked, optionally with a linking sequence. Generally, the linkingsequence is selected to provide flexibility (e.g., no defined secondarystructure other than a potential loop), for example, using a sequenceconsisting predominately or essentially of Gly, Ser, and/or Ala. In someembodiments, the linking amino acid sequence is from about 2 to about 25amino acids in length, which may form a loop. The circular permutant mayfurther comprise from 1 to about 30, or from about 1 to about 20, orfrom 1 to about 10, or from 1 to about 5 amino acid substitutions,deletions, or insertions with respect to the corresponding position ofthe parent or wild-type enzyme (e.g., based on the highest score localalignment). In some embodiments, the natural N-terminal Met ismaintained at its new position in the molecule, or in other embodimentsis deleted.

In some embodiments, at least one UGT enzyme is a chimeric UGT enzyme,in which the N-terminal domain of one UGT is combined with theC-terminal domain of a different UGT enzyme. For example, the N-terminaland C-terminal domains are of two different enzymes selected from Table9, and each domain may further comprise from one to ten amino acidsubstitutions, deletions, and/or insertions relative to the parentdomain sequence. UGTs have two domains, a more variable N-terminalsubstrate binding (sugar acceptor) domain and a more conservedC-terminal UDP-glucose binding (sugar donor) domain. The N-terminaldomain is mostly determinant of substrate specificity for the enzyme,but some specificity is controlled by the C-terminal domain. Each ofthese domains makes up roughly half of the protein.

In some embodiments, the UGT enzyme having 1-2′ glycosylating activityis OsUGT1-2 (SEQ ID NO:7), SrUGT91D2 (SEQ ID NO:5), SrUGT91D1 (SEQ IDNO:4), SrUGT91D2e (SEQ ID NO:6) (see Table 9) or derivative thereof. Insome embodiments, the derivative has increased glycosylating activity atC19 of RebA. The UGT enzyme may generally have a level of identity thatis greater than about 50%, greater than about 60%, greater than about70%, greater than about 80%, greater than about 90%, or greater thanabout 95%, or greater than about 96, 97, 98, or 99% to one or more ofOsUGT1-2, SrUGT91D2, SrUGT91D1, and SrUGT91D2e.

The similarity or identity of nucleotide and amino acid sequences, i.e.the percentage of sequence identity, can be determined via sequencealignments. Such alignments can be carried out with several art-knownalgorithms, such as with the mathematical algorithm of Karlin andAltschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) orwith the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T.J. (1994) Nucleic Acids Res. 22, 4673-80). The grade of sequenceidentity (sequence matching) may be calculated using e.g. BLAST, BLAT orBlastZ (or BlastX). A similar algorithm is incorporated into the BLASTNand BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215: 403-410.BLAST polynucleotide searches can be performed with the BLASTN program,score=100, word length=12.

BLAST protein searches may be performed with the BLASTP program,score=50, word length=3. To obtain gapped alignments for comparativepurposes, Gapped BLAST is utilized as described in Altschul et al (1997)Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs are used.Sequence matching analysis may be supplemented by established homologymapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b,19 Suppl 1:154-162) or Markov random fields.

In some embodiments the UGT enzyme having 1-2′ glycosylating activity isOsUGT1-2 or derivative thereof, and which is optionally a circularpermutant of OsUGT1-2 comprising one or more amino acid substitutions,deletions, and/or insertions that increase 1-2′ glycosylating activityat C19 of RebA. For example, the 1-2′ glycosylating enzyme may have acut site that aligns with or corresponds to a position within aminoacids 190 to 210 of OsUGT1-2 (SEQ ID NO:7), and may be a position withinamino acids 194 to 200 of SEQ ID NO:7 in some embodiments, such asposition 195, 196, 197, or 199. The circular permutant may optionallyhave a linker sequence between the amino acids that correspond to theN-terminal and C-terminal residues of OsUGT1-2. The linker may vary inlength, such as in the range of 2 to about 25 amino acids. For example,the linker may be from about 8 to about 20 amino acids in length, suchas about 17 amino acids in some embodiments. In some embodiments, thecircular permutant does not contain any linking sequence. The circularpermutant may further contain from 1 to 20, or from 1 to 10, or from 1to 5 amino acid substitutions, additions, or deletions from thewild-type sequence (determined by local alignment of the mutatedsequence to OsUGT1-2). In some embodiments, an Ala is inserted orsubstituted at position 2 to decrease enzyme turnover in the cell. Insome embodiments, the mutations collectively increase 1-2′ glycosylatingactivity at C19 of RebA (e.g., when evaluated at 22° C., 27° C., or 30°C.).

In some embodiments, the UGT enzyme having 1-2′ glycosylating activityis a circular permutant of OsUGT1-2, with a cut-site corresponding toposition 195, 196, 197, 198, or 199 of OsUGT1-2. An exemplary circularpermutant, named MbUGT1-2, is disclosed herein. The circular permutantmay have amino acid substitutions at one or more of positionscorresponding to positions 14, 16, 89, 185, 365, 366, 395, 396, 417,420, 421, 422, 424, 427, 428, 430, 431, 432, 434 and/or 463 of thewild-type enzyme. In some embodiments, the circular permutant has anamino acid substitution at position 14, and such substitution may be anaromatic amino acid, such as Trp or Tyr. In these or other embodiments,the circular permutant has an amino acid substitution at position 366,and the substituted amino acid is optionally Pro. In these or otherembodiments, the circular permutant has an amino acid substitution atposition 420, and the substituted amino acid is optionally Glu. In theseor other embodiments, the circular permutant has an amino acidsubstitution at position 421, and the substituted amino acid isoptionally Phe. In these or other embodiments, the circular permutanthas an amino acid substitution at position 424, and the substitutedamino acid is optionally Asp. In these or other embodiments, thecircular permutant has an amino acid substitution at position 427, andthe substituted amino acid is optionally Glu. In these or otherembodiments, the circular permutant has an amino acid substitution atposition 428, and the substituted amino acid is optionally Glu. In theseor other embodiments, the circular permutant has an amino acidsubstitution at position 432, and the substituted amino acid isoptionally Tyr, His, Trp, Asp, or Glu. In some embodiments, the enzymecontains an insertion of from 2-5 amino acids between amino acids 424and 427, such as the sequence Gly-Pro-Ser. In some embodiments, the UGThaving 1-2′ glycosylating activity comprises the amino acid sequence ofSEQ ID NO:9 (MbUGT1-2), or an enzyme having at least about 50% identity,at least about 60% identity, at least about 70% identity, at least about80% identity, at least about 85% identity, or at least about 90%identity, or at least about 95% identity, or at least 96%, 97%, 98% or99% identity to SEQ ID NO:9, and having 1-2′ glycosylating activity atone or more of C19 of RebA or C13 of steviolmonoside.

In some embodiments, the UGT enzyme having 1-2′ glycosylating activityis a circular permutant of OsUGT1-2, with a cut site corresponding toposition 196 of OsUGT1-2. An exemplary circular permutant, namedMbUGT1,2-2 (SEQ ID NO:45), is disclosed herein. The circular permutanthas amino acid substitutions at one or more of positions 16, 422, 430,and 434 of the wild-type enzyme. In some embodiments, the circularpermutant has an amino acid substitution at position 16, and suchsubstitution may be an aromatic amino acid, such as Trp. In these orother embodiments, the circular permutant has an amino acid substitutionat position 422, and the substituted amino acid is optionally Glu. Inthese or other embodiments, the circular permutant has an amino acidsubstitution at position 430, and the substituted amino acid isoptionally Glu. In these or other embodiments, the circular permutanthas an amino acid substitution at position 434, and the substitutedamino acid is optionally His. In some embodiments, the enzyme does notcontain any linking sequence between the natural N- and C-termini aminoacids, and the natural N-terminal Met may be optionally deleted. In someembodiments, the UGT having 1-2′ glycosylating activity comprises theamino acid sequence of SEQ ID NO:45, or an enzyme having at least about50% identity, at least about 60% identity, at least about 70% identity,at least about 80% identity, at least about 85% identity, or at leastabout 90% identity, or at least about 95% identity, or at least 96%,97%, 98% or 99% identity to SEQ ID NO:45, and having 1-2′ glycosylatingactivity at one or more of C19 of RebA or C13 of steviolmonoside.

In some embodiments, the UGT enzyme having 1-3′ glycosylating activityis SrUGT76G1, or derivative thereof having the same or increasedglycosylation activity at C19 of RebD or C13 of stevioside. In someembodiments, the UGT enzyme having 1-3′ glycosylating activity is aderivative of SrUGT76G1 that includes an amino acid substitution at oneor more of positions 77, 78, 81, 82, 93, 94, 155, 192, 200, 202, 205,283, 284, 379, and 397 of SEQ ID NO: 3 (see Table 13). In someembodiments, the derivative has an amino acid substitution at positionL200 (numbered according to the wild type enzyme), and which isoptionally Ala or Gly. In these embodiments, the derivative may furtherhave an amino acid substitution at position 284 (e.g., Ala) and/or 379(e.g., Gly), and/or 192 (e.g., Ala). In some embodiments, an Ala isinserted or substituted at position 2 to decrease turnover in the cell.In some embodiments, the UGT enzyme has at least about 80% identity, atleast about 85% identity, at least about 90% identity, or at least about95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3, with the provisothat the amino acid corresponding to position 200 of SEQ ID NO:3 is Alaor Gly. As shown in Table 13, the substitution of L200A or L200Gprovides for large improvements in activity at both C19 and C13.

Additional modification to UGT76G1 include modification at one or moreof positions 22, 25, 145, 154, 256, and 282, such as one or more ofQ22G, Q22H, I25F, 125W, T145A, T145G, T145P, H154R, L256P, L256W, L256T,L256G, L256A, L256R, L256E, S281G and S282N. These modifications aredisclosed in WO 2014/122227, which is hereby incorporated by reference.In some embodiments, these additional modifications to UGT76G1 exhibitsuperior properties in combination with the modifications at positions77, 78, 81, 82, 83, 93, 94, 155, 192, 200, 202, 205, 283, 284, 378, 379,and 397.

In some embodiments, the UGT enzyme having 1-3′ glycosylating activityis a circular permutant of SrUGT76G1, with a cut-site corresponding to aposition within amino acids 170 to 290 (e.g, 190-210, 196-200 or260-280) of SrUGT76G1. In some embodiments, the cut site corresponds toposition 196 or 264 of the wild-type enzyme. The circular permutant(e.g., MbUGT1-3), may have from 1 to 30, or from 1 to 20, or from 1 to10, or from 1 to 5 amino acid substitutions, deletions, and/orinsertions with respect to the corresponding position of the wild-typesequence. In some embodiments, the UGT having 1-3′ glycosylatingactivity comprises the amino acid sequence of SEQ ID NO: 10 (MbUGT1-3),or comprises an amino acid sequence having at least about 50% identity,at least about 60% identity, at least about 70% identity, at least about80% identity, at least about 85% identity, or at least about 90%identity, or at least about 95% identity, or at least 96%, 97%, 98% or99% identity to SEQ ID NO: 10, and having 1-3′ glycosylating activity atone or more of C19 of RebD or C13 of stevioside. In some embodiments,Ala is substituted or inserted at position 2 to decrease turnover in thecell.

In various embodiments, the host cell or method of the invention furtherinvolves a UGT enzyme that converts steviol to steviolmonoside. In someembodiments, the UGT enzyme that converts steviol to steviolmonoside isSrUGT85C2, or derivative thereof. In some embodiments, the enzymecontains from 1 to about 50, or from 1 to about 20, or from 1 to about10 amino acid substitutions, deletions, and/or insertions with respectto SEQ ID NO: 1. For example, the derivative may have at least about 65%identity, or at least about 70% identity, or at least about 80% identityto SEQ ID NO: 1, or at least 90% identity to SEQ ID NO: 1, or at least95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1, while maintainingthe same or similar activity for converting steviol to steviolmonoside(e.g., in vitro or in vivo). Exemplary amino acid modifications areshown in Table 11. In some embodiments, the enzyme that converts steviolto steviolmonoside is a derivative of SrUGT85C2 having an amino acidsubstitution at position 215 of the wild type enzyme. In someembodiments, the amino acid at the position corresponding to 215 of thewild type enzyme is threonine, serine, glycine, or alanine (the wildtype amino acid is Proline). In some embodiments, the amino acid at saidposition 215 is threonine. In these or other embodiments, the derivativeof SrUGT85C2 has a mutation at one or more of positions 308, 311, 316,349, and/or 414 (numbered in accordance with the wild type enzyme. Insome embodiments, the amino acid at position 308 is threonine, and/orthe amino acid at position 311 is glutamine, and/or the amino acid atposition 316 is alanine, and/or the amino acid at position 349 isglutamic acid, and/or the amino acid at position 414 is glycine. In someembodiments, an Ala is inserted or substituted at the second position tolimit turnover in the cell.

In various embodiments, the host cell or method further involves a UGTenzyme that converts steviolbioside to stevioside, which in someembodiments is SrUGT74G1, or derivative thereof. In some embodiments,the enzyme contains from 1 to about 50, or from 1 to about 20, or from 1to 10 amino acid substitutions, deletions, and/or insertions withrespect to SEQ ID NO: 2. For example, the derivative may have at least80% identity to SEQ ID NO: 2, at least 90% identity to SEQ ID NO: 2, orat least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2, whilemaintaining the same or similar activity for converting steviolbiosideto stevioside (e.g., in vitro or in vivo).

In some embodiments, the UGT enzyme that converts steviolbioside tostevioside is a circular permutant of SrUGT74G1 (e.g., MbUGTC19). Insome embodiments, the circular permutant has a cut site corresponding toan amino acid within positions 180 to 280 (e.g., 250 to 270) ofSrUGT74G1. The circular permutant may have a linking sequence betweenthe original N- and C-termini of from 1 to 10 amino acids (e.g., GSG).The circular permutant may have from 1 to 30, or from 1 to 20, or from 1to 10, or from 1 to 5 amino acid substitutions, deletions, and/orinsertions with respect to the corresponding position of the wild-typesequence. In some embodiments, the SrUGT74G1 circular permutantcomprises the amino acid sequence of SEQ ID NO: 8 (MbUGTC19) or SEQ IDNO: 46 (MbUGTC19-2), or comprises an amino acid sequence having at leastabout 50% identity, at least about 60% identity, at least about 70%identity, at least about 80% identity, at least about 85% identity, orat least about 90% identity, or at least about 95% identity, or at least96%, 97%, 98% or 99% identity to SEQ ID NO: 8 or 46, and having activityfor converting steviolbioside to stevioside.

In some embodiments, the host cell produces steviol substrate throughone or more pathway modules comprising a kaurene synthase (KS), kaureneoxidase (KO), and a kaurenoic acid hydroxylase (KAH), the host cellfurther comprising a cytochrome P450 reductase (CPR) for regeneratingone or more of the KO and KAH enzymes. In some embodiments, the KAH isKAH of Stevia rebaudiana, Arabidopsis thaliana, Vitis vinifera, orMedicago trunculata, or a derivative thereof (e.g., having at least 80%,or at least 90%, or at least 95%, or at least 97% sequence identity tothe wild type sequence). In some embodiments, the KAH is an Arabidopsisthaliana KAH (AtKAH), or derivative thereof. The AtKAH may have one ormore amino acid substitutions, insertions, and/or deletions thatincrease the rate of kaurenoic acid hydroxylase activity or otherwiseimprove enzyme productivity or expression, including for example anN-terminus engineered for functional expression in E. coli. In someembodiments, the AtKAH has an amino acid substitution at one or morepositions (e.g., two-ten positions) of the parent sequence of SEQ IDNO:29 as shown in Table 6 that increases production of steviol orkaurenoic acid. Exemplary substitutions include substitutionscorresponding to the following positions of SEQ ID NO:29: 25 (e.g.,A25L), 79 (e.g., S79T), 119 (e.g., T119C), 137 (e.g., I137L), 142 (e.g.,I142V), 155 (e.g., R155K), 180 (e.g., M180L), 193 (e.g., E193G), 196(e.g. C196A), 197 (e.g., D197E), 226 (A226E), 235 (e.g., L235Q), 238(e.g., I238M), 245 (F245L, F245V), 272 (e.g., L272I), 285 (e.g., I285R),287 (e.g., C287S), 325 (e.g., C325I, C325M), 330 (e.g., F330L), 334(e.g., D334E), 339 (e.g., S339T), 352 (e.g., S352E), 373 (e.g., E373D),397 (e.g., I397F), 470 (e.g., V470L), 499 (e.g., Q499V), 506 (e.g.,L506M), 507 (e.g., L507I, L507T, L507V). In some embodiments, the AtKAHenzyme is a derivative having an amino acid substitution at position 331(with respect to the wild type sequence), which in some embodiments,improves the productivity of the enzyme at higher temperatures (e.g.,higher than 22° C.). In some embodiments, the amino acid at position 331is Ile.

N-terminal modifications to achieve functional expression of the P450enzyme SrKO are illustrated in FIG. 9. These modifications or similarmodifications may be made to achieve functional expression of KAH,including AtKAH. For example, all or portions of the transmembraneregion may be deleted, such as from 4 amino acids to about 39 aminoacids, or in some embodiments, from about 6 amino acids to about 25amino acids, or about 4 to about 20 amino acids, or about 29 aminoacids, or about 39 amino acids. The deletions are preferably taken fromthe N-terminal portion of the transmembrane region. This portion isreplaced with a solubilization tag of from about 4 to about 20 aminoacid residues, such as from about 4 to about 12 residues (e.g., eightamino acid residues). The tag is constructed predominantly ofhydrophobic amino acids, which are optionally selected from Ala, Leu,Ile, Val, and Phe. An exemplary sequence for the functional expressionis the N-terminal tag: MALLLAVF (SEQ ID NO: 47). In some embodiments,the AtKAH has a truncation of 14 amino acids, with the addition of theN-terminal tag (e.g., SEQ ID NO: 29), optionally having the substitutionC331I (position nomenclature based on the wild type enzyme).

Alternative N-terminal tag sequences for P450 enzymes are described inProvisional Application No. 62/208,166, filed Aug. 21, 2015, and whichfind use in certain embodiments of the present invention. For example,the transmembrane domain (or “N-terminal anchor”) can be derived from anE. coli gene selected from waaA, ypfN, yhcB, yhbM, yhhm, zipA, ycgG,dj1A, sohB, lpxK, F11O, motA, htpx, pgaC, ygdD, hemr, and ycls. Thesegenes were identified as inner membrane, cytoplasmic C-terminus proteinsthrough bioinformatic prediction as well as experimental validation. Theinvention may employ an N-terminal anchor sequence that is a derivativeof the E. coli wild-type transmembrane domain, that is, having one ormore mutations with respect to the wild-type sequence. In exemplaryembodiments, the membrane anchor sequence is from about 8 to about 75amino acids in length. For example, the membrane anchor may be fromabout 15 to about 50, or from about 15 to about 40, or from about 15 toabout 30, or from about 20 to about 40, or from about 20 to about 30amino acids in length.

In some embodiments, the Kaurene Synthase (KS) is from Steviarebaudiana, Zea mays, Populus trichocarpa, Arabidopsis thaliana, Erwinatracheiphila or derivative thereof (e.g., having at least 80%, or atleast 90%, or at least 95%, or at least 97% sequence identity to thewild type sequence). Further, the cell may express a copalyl diphosphatesynthase (CPPS) from Stevia rebaudiana, Streptomyces clavuligerus,Bradyrhizobium japonicum, Zea mays, Arabidopsis thaliana, Erwinatracheiphila, or derivative thereof (e.g., having at least 80%, or atleast 90%, or at least 95%, or at least 97% sequence identity to thewild type sequence). In some embodiments, the host cell expresses abifunctional CPPS and KS enzyme, which is optionally selected fromPhomopsis amygdali, Physcomitrella patens, Gibberella fujikuroi enzyme,or derivative thereof. Such derivative may generally have at least about75%, or at least about 80%, or at least about 85%, or at least about90%, or at least about 95%, or at least 96%, 97%, 98%, or 99% identityto the parent sequence (e.g., see Table 1). In some embodiments, thecell expresses Erwina tracheiphila CPPS and KS enzymes, or derivativesthereof.

In some embodiments, the host cell expresses a Kaurene Oxidase fromStevia rebaudiana (SrKO), Arabidopsis thaliana, Gibberella fujikoroi, orTrametes versicolor, or a derivative thereof, which is optionallymodified at the N-terminus for functional expression in E. coli (asdescribed above and as shown in FIG. 9). In some embodiments, the CPR isan enzyme of Stevia rebaudiana (SrCPR), Arabidopsis thaliana, orGiberella fujikuroi, or a derivative thereof, which is optionallymodified at the N-terminus for functional expression in E. coli.

The SrKO may have one or more amino acid modifications that improve itsactivity. Exemplary modifications are disclosed in U.S. ProvisionalApplication No. 62/040,284, which is hereby incorporated by reference inits entirety. For example, the SrKO may comprise one or more amino acidmodifications at positions (with respect to SEQ ID NO:22: 47 (e.g.,L47I), 59 (e.g., Y59H), 60 (e.g., M60K), 63 (e.g., T63A), 67 (e.g.,A67E), 76 (e.g., K76R), 80 (e.g., T80C), 82 (e.g., M82V), 85 (e.g.,V85L, V85I), 86 (e.g., S86N), 100 (e.g., Q100S), 106 (e.g., N106K), 112(e.g., K112T), 116 (A116R), 119 (e.g., T119S), 123 (e.g., M123T, M123Q,M123F, M123T), 127 (e.g., D127G), 129 (e.g., Y129F), 140 (e.g., A140R),149 (e.g., K149R), 150 (e.g., H150F), 171 (e.g., N171D), 180 (e.g.,L180F), 183 (e.g., I183V), 208 (e.g., D208E), 232 (e.g., D232E), 267(e.g., S267A), 272 (e.g., H272Q), 284 (e.g., S284C), 286 (e.g., I286L),294 (e.g., Q294K), 299 (e.g., Q299E), 310 (e.g., I310T, I310V), 371(e.g., R371K, R371I), 375 (e.g., V375T, V375I, V375L), 378 (e.g.,I378V), 382 (e.g., H382Y), 388 (e.g., V388Q, V388M), 393 (e.g., H393D),400 (e.g., L400I), 413 (e.g., V413K, V413D), 434 (e.g., F434L), 442(e.g., G442A), 450 (e.g., S450A), 454 (e.g., L454M), 460 (e.g., G460A),464 (e.g., M464L), 475 (e.g., M475G), 487 (e.g., T487N), 492 (e.g.,P492K), and 497 (e.g., I497L). In some embodiments, the SrKO contains atruncation of about 20 amino acids of the N-terminal transmembranedomain, with addition of an N-terminal tag sequence (described above).The SrKO may contain an Ala at the 2nd position to decrease enzymeturnover in the cell.

In some embodiments, the P450 reductase partner(s) include Steviarebaudiana (Sr)CPR, Stevia rebaudiana (Sr)CPR1, Arabidopsis thaliana(At)CPR, Taxus cuspidata (Tc)CPR, Artemisia annua (Aa)CPR, Arabidopsisthaliana (At)CPR1, Arabidopsis thaliana (At)CPR2, Arabidopsis thaliana(At)R2, Stevia rebaudiana (Sr)CPR2, Stevia rebaudiana (Sr)CPR3, orPelargonium graveolens (Pg)CPR. Any of these P450s can be derivatized insome embodiments, for example, to introduce from 1 to about 20mutations, or from about 1 to about 10 mutations. These CPR proteins arefurther described in PCT/US15/46369, which disclosure is herebyincorporated by reference.

In some embodiments, the host cell is an E. coli that contains a singleCPR enzyme (e.g., SrCPR), and which is chromosomally integrated, andsupports both the SrKO and AtKAH enzymes, for example.

In some embodiments, the host cell expresses a geranylgeranylpyrophosphate synthase (GGPPS), which is optionally of Taxus canadensis,Abies grandis, Aspergillus nidulans, Stevia rebaudiana, Gibberellafujikuroi, Mus musculus, Thalassiosira pseudonana, Streptomycesmelanosporofaciens, Streptomyces clavuligerus, Sulfulubusacidocaldarius, Synechococcus sp. (e.g., JA-3-3Ab), Arabidopsisthaliana, Marine bacterium 443, Paracoccus haeundaensis, Chlorobiumtepidum TLS, Synechocystis sp. (PCC 6803), Thermotoga maritima HB8,Corynebacterium glutamicum, Therms thermophillus HB27, Pyrobaculumcalidifontis JCM 11548, or derivative thereof. See Table 1. Suchderivative may generally have at least about 60%, or at least about 70%,or at least about 75%, or at least about 80%, or at least about 85%, orat least about 90%, or at least about 95%, or at least 96%, 97%, 98%, or99% identity to the parent sequence (e.g., see Table 1). In someembodiments, the GGPPS is Taxus canadensis or derivative thereof. Insome embodiments, the Taxus GGPPS is an N-terminal truncated sequence(e.g., with the N-terminal 70 to 110, such as about 98, amino acidstruncated). The truncated sequence may further comprise from about 1 toabout 10, such as from about 1 to about 5 amino acid substitutions,deletions, and/or insertions at the corresponding wild-type sequence. Anexemplary truncated sequence is disclosed herein as SEQ ID NO: 12. Insome embodiments, the GGPPS is from Cornybacterium glutamicum orderivative thereof, which can provide advantages in productivity attemperatures higher than 22° C.

In some embodiments, the host cell expresses a pathway producingiso-pentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP).In some embodiments, the pathway is a methylerythritol phosphate (MEP)pathway and/or a mevalonic acid (MVA) pathway.

The MEP (2-C-methyl-D-erythritol 4-phosphate) pathway, also called theMEP/DOXP (2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose5-phosphate) pathway or the non-mevalonate pathway or the mevalonicacid-independent pathway refers to the pathway that convertsglyceraldehyde-3-phosphate and pyruvate to IPP and DMAPP. The pathwaytypically involves action of the following enzymes:1-deoxy-D-xylulose-5-phosphate synthase (Dxs),1-deoxy-D-xylulose-5-phosphate reductoisomerase (IspC),4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD),4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE),2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF),1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), andisopentenyl diphosphate isomerase (IspH). The MEP pathway, and the genesand enzymes that make up the MEP pathway, are described in U.S. Pat. No.8,512,988, which is hereby incorporated by reference in its entirety.For example, genes that make up the MEP pathway include dxs, ispC, ispD,ispE, ispF, ispG, ispH, idi, and ispA. In some embodiments, steviol isproduced at least in part by metabolic flux through an MEP pathway, andwherein the host cell has at least one additional copy of a dxs, ispD,ispF, and/or idi gene. As disclosed in U.S. Pat. No. 8,512,988, thelevel of the metabolite indole can be used as a surrogate marker forefficient production of terpenoid products in E. coli through the MEPpathway.

The MVA pathway refers to the biosynthetic pathway that convertsacetyl-CoA to IPP. The mevalonate pathway typically comprises enzymesthat catalyze the following steps: (a) condensing two molecules ofacetyl-CoA to acetoacetyl-CoA (e.g., by action of acetoacetyl-CoAthiolase); (b) condensing acetoacetyl-CoA with acetyl-CoA to formhydroxymethylglutaryl-CoenzymeA (HMG-CoA) (e.g., by action of HMG-CoAsynthase (HMGS)); (c) converting HMG-CoA to mevalonate (e.g., by actionof HMG-CoA reductase (HMGR)); (d) phosphorylating mevalonate tomevalonate 5-phosphate (e.g., by action of mevalonate kinase (MK)); (e)converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate (e.g.,by action of phosphomevalonate kinase (PMK)); and (f) convertingmevalonate 5-pyrophosphate to isopentenyl pyrophosphate (e.g., by actionof mevalonate pyrophosphate decarboxylase (MPD)). The MVA pathway, andthe genes and enzymes that make up the MEP pathway, are described inU.S. Pat. No. 7,667,017, which is hereby incorporated by reference inits entirety.

The host cell may be prokaryotic or eukaryotic. For example, the hostcell may be a bacteria selected from E. coli, Bacillus subtillus, orPseudomonas putida. In some embodiments, the host cell is a species ofSaccharomyces, Pichia, or Yarrowia, including Saccharomyces cerevisiae,Pichia pastoris, and Yarrowia lipolytica. The host cell may be an E.coli having a duplication or overexpression of dxs, idi, IspD, and IspFincreasing production of IPP and DMAPP.

In some embodiments, the host cell is an E. coli having one or moregenetic modifications increasing the production of UDP-glucose, forexample, increasing UDP-glucose substrate availability. To improveavailability of UDP-glucose for steviol glycosylation, a series of geneknock-outs and gene insertions can be introduced to increase carbon fluxto UDP-glucose and decrease flux in pathways away from UDP-glucose(e.g., glycogen synthesis and carbon storage). For example, geneticmodifications can increase importation of sucrose into the cell andsplit it into fructose and glucose via the activity of sucrosephosphorylase. A subsequent series of knock-outs can alter primarymetabolism so as to force biomass to be synthesized using only fructoseas carbon source, leaving glucose to be funneled exclusively towardsUDP-glucose biosynthesis. Exemplary modifications to an E. coli strainto enact this strategy are listed in Table 7. Modifications are furtherdescribed in PCT/EP2011/061891, which is hereby incorporated byreference in its entirety. In some embodiments, the one or more geneticmodifications include ΔgalE, ΔgalT, ΔgalK, ΔgalM, ΔushA, Δagp, Δpgm,duplication of E. coli GALU, and expression of Bacillus substillus UGPA,BaSP.

In an exemplary embodiment, the host cell is an E. coli that comprisesthe following heterologously expressed genes: Taxus canadensis GGPPS orderivative thereof, Phaeosphaeria sp. PsCK or derivative thereof, Steviarebaudiana KO or derivative thereof, Arabidopsis thaliana KAH orderivative thereof, Stevia rebaudiana CPR or derivative thereof (andwhich is the only CPR enzyme expressed by the host cell), Steviarebaudiana UGT85C2 or derivative thereof, Stevia rebaudiana UGT74G1 ofderivative thereof, Stevia rebaudiana UGT76G1 or derivative thereof, andMbUGT1-2 or derivative thereof. Various derivatives of these enzymes aredisclosed herein. In some embodiments, the E. coli contains apolycistronic expression module of KAH-KO, and contains a single copy ofSrCPR (or derivative) that is chromosomally integrated. In someembodiments, the E. coli is modified to increase availability ofUDP-glucose as described above. In some embodiments, the E. coli has anadditional copy of dxs, idi, ispD, and ispF genes. In some embodiments,one or more expressed proteins contain an Alanine at position 2, toprovide additional stability in vivo.

In other embodiments, the host cell is an E. coli that comprises thefollowing heterologously expressed genes: Cornybacterium glutamicumGGPPS or derivative thereof; Erwina tracheiphila CPPS and KS orderivative of one or both; Stevia rebaudiana KO or derivative thereof;Arabidopsis thaliana KAH or derivative thereof; a Stevia rebaudiana CPRor derivative thereof; Stevia rebaudiana UGT85C2 or MbUGTc13 orderivative thereof; Stevia rebaudiana UGT74G1 or derivative thereof (orMbUGTC19, MbUGTC19-2, or derivative thereof); Stevia rebaudiana UGT76G1or derivative thereof (or MbUGT1-3 of derivative thereof); and OsUGT1-2,SrUGT91D2, or derivative thereof, or MbUGT1-2 or MbUGT1,1-2 orderivative thereof. Various derivatives of these enzymes are disclosedherein. In some embodiments, the E. coli contains a polycistronicexpression module of KAH-KO, and contains a single copy of SrCPR that ischromosomally integrated. In some embodiments, the E. coli is modifiedto increase availability of UDP-glucose as described above. In someembodiments, the E. coli has an additional copy of one or more (or all)of dxs, idi, ispD, and ispF genes. In some embodiments, one or moreexpressed proteins contain an Alanine at position 2, to provideadditional stability in vivo. In some embodiments, the E. coli providesincreased productivity of Reb M or Reb D at temperatures above about 24°C., such as about 27° C. or more, or about 30° C. or more, or about 32°C. or more, or about 34° C. or more, or about 37° C.

In some embodiments, the method further comprises recovering the desiredsteviol glycoside(s) (e.g., RebM or RebD) from culture media. In someembodiments, the desired steviol glycoside (e.g., RebM or RebD) isproduced in the culture media at a concentration of at least about 10mg/L, or at least about 100 mg/L, or at least about 200 mg/L, or atleast about 500 mg/L, or at least about 1 g/L, or at least about 10 g/L.

Optionally, the method of the present invention further comprisesseparating the target steviol glycoside from the starting composition.The target steviol glycoside can be separated by any suitable method,such as, for example, crystallization, separation by membranes,centrifugation, extraction, chromatographic separation or a combinationof such methods. Fractions containing different glycoside fractions canbe blended to prepare defined products. Alternatively, RebM and RebD,for example, can be prepared and purified from separate cultures, andblended at a predetermined ratio.

In another aspect, the invention provides a method for production ofsteviol glycosides having at least 4 glycosylations in E. coli. Inaccordance with the invention, the E. coli cell comprises a plurality ofUGT enzymes, which may include one or more enzymes described herein,that together perform at least 4, at least 5, or at least 6 (including 7or 8), sequential glycosylation reactions. As disclosed herein, theglycosylation substrates and lower glycosylation products accumulate inthe E. coli cell sufficiently to allow downstream reactions to proceedat an acceptable rate, with a high majority of the glycosylationproducts ultimately accumulating extracellularly, most likely throughthe action of a membrane transporter. The steviol glycosides can bepurified from media components. Thus, in some embodiments, the methodscomprise separating growth media from the E. coli cells, for exampleusing batch, continuous, or semi-continuous bioreactor processes, andisolating the desired glycosylation products (e.g, Reb M) from thegrowth media.

In still other aspects, the invention provides methods for production ofsteviol glycosides (including Reb D, Reb M, Reb E, Reb I and otherglycosylation products) in E. coli. Generally, the desired steviolglycoside has at least 2 glycosylations, such as 2, 3, 4, 5, 6, 7, or 8glycosylations. In some embodiments, the steviol glycoside is RebM orRebD. While many of the enzymes known for production of steviol in hostcells are plant enzymes, which often have optimal temperatures in therange of 20-24° C., E. coli growth rate and metabolism are optimal athigher temperatures. The present disclosure enables production ofsteviol glycosides at high yield in E. coli, by enabling enzymeproductivity at temperatures above 24° C., such as from 24° C. to 37°C., or from 27° C. to 37° C., or from 30° C. to 37° C.

While commercial biosynthesis in E. coli can often be limited by thetemperature at which overexpressed and/or foreign enzymes are stable,the present disclosure in some embodiments allows for cultures to bemaintained at higher temperatures, resulting in higher yields and higheroverall productivity. In some embodiments, the culturing is conducted atabout 30° C. or greater, or about 31° C. or greater, or about 32° C. orgreater, or about 33° C. or greater, or about 34° C. or greater, orabout 35° C. or greater, or about 36° C. or greater, or about 37° C.

The host cells and methods are further suitable for commercialproduction of steviol glycosides, that is, the cells and methods can beproductive at commercial scale. In some embodiments, the size of theculture is at least about 100 L, at least about 200 L, at least about500 L, at least about 1,000 L, or at least about 10,000 L. In anembodiment, the culturing may be conducted in batch culture, continuousculture, or semi-continuous culture.

In some aspects, the invention provides methods for making a productcomprising a steviol glycoside ingredient, which is RebM or RebD in someembodiments. The method comprises culturing a strain described hereinthat produces the steviol glycoside, recovering the steviol glycoside,and incorporating the steviol glycoside into a product, such as a food,beverage, oral care product, sweetener, flavoring agent, or otherproduct.

Purified steviol glycosides, prepared in accordance with the presentinvention, may be used in a variety of products including, but notlimited to, foods, beverages, texturants (e.g., starches, fibers, gums,fats and fat mimetics, and emulsifiers), pharmaceutical compositions,tobacco products, nutraceutical compositions, oral hygiene compositions,and cosmetic compositions. Non-limiting examples of flavors for whichRebM can be used in combination include lime, lemon, orange, fruit,banana, grape, pear, pineapple, mango, bitter almond, cola, cinnamon,sugar, cotton candy and vanilla flavors. Non-limiting examples of otherfood ingredients include flavors, acidulants, and amino acids, coloringagents, bulking agents, modified starches, gums, texturizers,preservatives, antioxidants, emulsifiers, stabilizers, thickeners andgelling agents.

Highly purified target steviol glycoside(s) obtained according to thisinvention may be incorporated as a high intensity natural sweetener infoodstuffs, beverages, pharmaceutical compositions, cosmetics, chewinggums, table top products, cereals, dairy products, toothpastes and otheroral cavity compositions, etc.

Highly purified target steviol glycoside(s) obtained according to thisinvention can be used in combination with various physiologically activesubstances or functional ingredients. Functional ingredients generallyare classified into categories such as carotenoids, dietary fiber, fattyacids, saponins, antioxidants, nutraceuticals, flavonoids,isothiocyanates, phenols, plant sterols and stanols (phytosterols andphytostanols); polyols; prebiotics, probiotics; phytoestrogens; soyprotein; sulfides/thiols; amino acids; proteins; vitamins; and minerals.Functional ingredients also may be classified based on their healthbenefits, such as cardiovascular, cholesterol-reducing, andanti-inflammatory.

Highly purified target steviol glycoside(s) obtained according to thisinvention may be applied as a high intensity sweetener to produce zerocalorie, reduced calorie or diabetic beverages and food products withimproved taste characteristics. It may also be used in drinks,foodstuffs, pharmaceuticals, and other products in which sugar cannot beused. In addition, highly purified target steviol glycoside(s),particularly, RebM can be used as a sweetener not only for drinks,foodstuffs, and other products dedicated for human consumption, but alsoin animal feed and fodder with improved characteristics.

Examples of products in which highly purified target steviolglycoside(s) may be used as a sweetening compound include, but are notlimited to, alcoholic beverages such as vodka, wine, beer, liquor, andsake, etc.; natural juices; refreshing drinks; carbonated soft drinks;diet drinks; zero calorie drinks; reduced calorie drinks and foods;yogurt drinks; instant juices; instant coffee; powdered types of instantbeverages; canned products; syrups; fermented soybean paste; soy sauce;vinegar; dressings; mayonnaise; ketchups; curry; soup; instant bouillon;powdered soy sauce; powdered vinegar; types of biscuits; rice biscuit;crackers; bread; chocolates; caramel; candy; chewing gum; jelly;pudding; preserved fruits and vegetables; fresh cream; jam; marmalade;flower paste; powdered milk; ice cream; sorbet; vegetables and fruitspacked in bottles; canned and boiled beans; meat and foods boiled insweetened sauce; agricultural vegetable food products; seafood; ham;sausage; fish ham; fish sausage; fish paste; deep fried fish products;dried seafood products; frozen food products; preserved seaweed;preserved meat; tobacco; medicinal products; and many others. Inprinciple it can have unlimited applications.

During the manufacturing of products such as foodstuffs, drinks,pharmaceuticals, cosmetics, table top products, and chewing gum, theconventional methods such as mixing, kneading, dissolution, pickling,permeation, percolation, sprinkling, atomizing, infusing and othermethods may be used.

EXAMPLES

Steviol glycosides are the natural constituents of the plant Steviarebaudiana, known commonly as Stevia. Steviol glycoside Rebaudioside M(RebM) (FIG. 1), whose taste profile drastically improves upon that ofother steviol glycosides, is an ideal candidate to replace currentlyused steviol glycosides such Rebaudioside A, but hasn't fulfilled thatpromise because of its low levels in the Stevia plant (<0.01%). Steviolis a diterpenoid that forms the core chemical structure of steviolglycosides like RebM (1).

Terpenoid biosynthesis has been engineered in both prokaryotic (e.g., E.coli) and eukaryotic (e.g., yeast) cells for heterologous production ofcomplex terpenoid molecules (2,3). The E. coli MEP-pathway isstoichiometrically superior and less byproduct accumulating compared tothe yeast MVA-pathway (4,5). A new metabolic engineering approach,multivariate modular metabolic engineering (MMME), and a platform E.coli strain capable of overproducing terpenoid precursors has beendescribed (4,6). MMME facilitates assessment and elimination ofregulatory and pathway bottlenecks by re-defining the metabolic networkas a collection of distinct modules (7). By grouping enzymes withsimilar turnovers into a subset, or module, and later equalizing theturnover of the different subsets by adjustingconcentrations/activities, one can maximize the ratio of pathwayturnover to resource expenditure.

MMME pathway engineering was applied in E. coli for the biosynthesis ofkaurene, the unfunctionalized terpene scaffold precursor for steviol andsteviol glycosides. Next, the downstream CYP450-mediated oxidationchemistry was engineered to demonstrate that the diterpenoid scaffoldsteviol can be biosynthesized in E. coli. Further, glycosylationchemistry for the conversion of steviol to steviol glycosides in E. coliwas engineered to develop a technology platform for producingglycosylated natural products. Further still, E. coli were engineered toproduce improved levels of UDP-glucose to support high levels of steviolglycoside production. This work provides for an economical,commercially-viable source for RebM (and other steviol glycosidesdescribed herein) in microbial systems from renewable resources.

Example 1: Biosynthesis of Steviol and Steviol Glycosides

Steviol glycosides are diterpenoid derivatives and their earlybiosynthetic pathways share common intermediates with gibberellic acidbiosynthesis (8). The overall linear pathway is modularized into fourparts: (1) the formation of starting precursor IPP and DMAPP from thecentral carbon metabolites derived from glucose, (2) the production ofthe first dedicated intermediate, kaurene; (3) biosynthesis of the keyintermediate, steviol; and (4) the formation of various steviolglycosides. A further module (5) is independently engineered to supportthe increased production of UDP-glucose, the second substrate necessaryfor glycosylation of steviol. The five modules are shown in FIG. 2.

In plants, the formation of common isoprenoid precursors IPP and DMAPPcan be derived from two biosynthetic routes; either the mevalonic acid(MVA) pathway or methylerythritol-phosphate (MEP) pathway (9). The firststep in steviol diterpenoid biosynthesis is conversion of IPP and DMAPPinto geranyl-geranyl diphosphate (GGPP). GGPP is the four subunitprecursor for all diterpenoid molecules. Next, protonation-initiatedcyclization of GGPP to copalyl diphosphate (CPP) is catalyzed by CPPsynthase (CPPS). Kaurene is then produced from CPP by anionization-dependant cyclization catalyzed by kaurene synthase (KS).These enzymes have been identified and characterized from the nativebiosynthetic pathway in Stevia. In addition to this, there arebi-functional enzymes characterized from the basal plant (Physcomitrellapatens) and fungal species (e.g., Gibberella fujikuroi and Phaeosphaeriasp.) for conversion of GGPP into kaurene (10,11). Kaurene is thenoxidized in a three-step reaction to kaurenoic acid, by kaurene oxidase(KO) a P450 mono-oxygenase. A full length KO cDNA was expressed in yeastand demonstrated that it could convert kaurene to kaurenoic acid (12).The next step in the pathway is the hydroxylation of kaurenoic acid bykaurenoic acid 13-hydroxylase (KAH). KAH, a cytochrome P450, wasexpressed in yeast and converted kaurenoic acid to steviol (13).

With the core steviol molecule assembled, a series of six glycosylationsattach six glucose moieties to the steviol core. The glycosyltransferaseenzymes (EC 2.4.1.17) responsible for these activities catalyze thetransfer of the glucose component of UDP-glucose to a small hydrophobicmolecule, in this case the steviol molecule (14). O-glycosylations occurat the C13 and C19 positions of steviol (FIG. 1), followed by 1-2′glycosylations and 1-3′ glycosylations at both these O-glucosyls toresult in six glycosylations in total. The order of glycosylations canbe quite complex, with various intermediate products forming givenvariation in the order of C13 or C19 glycosylations, as well as 1-2′ or1-3′ glycosylations (FIG. 3). Given the intermediate product poolsaccumulating in Stevia rebaudiana, a potential pathway for theproduction of RebM isSteviol>Steviolmonoside>Steviolbioside>Stevioside>RebaudiosideA>Rebaudioside D>Rebaudioside M. However, this does not preclude analternate pathway in microbial biosynthetic systems (FIG. 3).

Detailed understanding and characterization of biochemical pathways forsteviol glycosides and advancements in engineering of the upstreamisoprenoid pathway to reroute the IPP and DMAPP through heterologousbiosynthetic pathway engineering provides the basis for directed,sustainable production of purified and high quality steviol glycosidesin a convenient microbial-based bioprocess. The current plant-basedproduction and purification schemes present significant challenges toreducing costs. The microbial route described herein using plantpathways that have been reconstructed in microbial hosts offers superioropportunities for improving current processes and to generate superiorquality steviol glycosides that are of very low abundance in nature.

(A) Engineering Kaurene Biosynthesis in E. coli

Kaurene is the cyclic diterpenoid precursor for steviol and plant growthhormone gibberellic acid. The biosynthesis of kaurene consists of threesteps from the universal terpenoid precursor IPP and DMAPP. The threestep reaction from IPP and DMAPP is catalyzed by enzymes GPPS, CPS andKS or bifunctional CPPS/KS enzymes. The overall pathway up to kaurene isgrouped as two modules (FIG. 2). There have been several enzymes fromdifferent organisms characterized for the conversion of IPP and DMAPP toGGPP (15-18) and GGPP to kaurene (9-12,18) and kaurene to steviol(12,19-24) (Table 1). In higher plants, such as stevia, GGPP to kaurenebiosynthesis is carried out as two step reaction mediated by enzymescalled copalyl pyrophosphate synthase (CPPS) and kaurene synthase (KS).In the basal plant (Physcomitrella patens) and fungal (e.g., Gibberellafujikuroi and Phaeosphaeria sp.) species, the GGPP to kaurenebiosynthesis is carried out by bi-functional enzymes characterized inthese organisms. Similarly, there are multiple enzymes cloned andcharacterized as converting IPP and DMAPP to make GGPP. The first steptowards engineering kaurene biosynthesis is therefore selection ofenzymes. Enzymes from different species were selected to test forbiosynthesis of kaurene (Table 1). Studies on MMME optimization oftaxadiene biosynthesis show that the kinetics of TcGPPS are capable ofsupporting ˜1 g/L production of taxadiene and therefore otherditerpenes. To identify the best kaurene synthase ortholog, TcGPPS wasselected as the upstream candidate enzyme. Operons were then selectedcontaining KS-CPS-GGPPS (KCG) or bi-functional PsCK-GGPPS (CKG) to testthe pathway in the upstream pathway engineered strains. To modulate theexpression of the downstream kaurene pathway, the KS-CPPS-GGPPS (KCG)and CK-GGPPS (CKG) operons were cloned to a plasmid system with varyingcopy number and promoter strength (p5Trc, p10Trc, p20Trc and p5T7).Additionally, one copy of each kaurene operon was integrated into the E.coli chromosome under varying promoter strength, coupled with varyingupstream pathway expression levels.

Strains were selected with varying upstream and downstream expression tomodulate the pathway and test the productivity of the variouscombinations. These strains were subjected to small scale (2 mL) Hungatetube fermentation to characterize the phenotypic characteristics andkaurene productivity. As shown in FIG. 4, a complex non-linearaccumulation of kaurene was observed. Interestingly, KS from plantspecies (SrCPPS, SrKS and PpCK) showed similar profiles (FIG. 4A, B),whereas the pathways constructed with fungal enzymes (GfCK and PsCK)showed very similar patterns of product accumulation (FIG. 4C, D).Interestingly, the low-copy expression of pathways incorporating fungalenzymes showed relatively high productivity compared to the plant enzymepathways. The global maximum in product titer (˜140 mg/L) comes from aconstruct with exclusively plant enzymes (FIG. 4A, strain constructedwith Stevia rebaudiana genes with upstream under Trc promoter anddownstream in plasmid p20Trc). However, the completelychromosomally-integrated fungal pathway enzyme (PsCK) (FIG. 4D, strainwith upstream Trc and downstream T7-PsCKG) produced ˜100 mg/L ofkaurene. Comparing the expression of the downstream components of thesetwo strains, the Ch1T7PsCKG pathway is 23-fold less (1.5 a.u.) comparedto the p20TrcSrKCG (35 a.u.) under the same upstream pathway Ch1TrcMEPstrength (4). The key performance driver of a multistep/multi-modulepathway is optimal balance in the flux. Here in the strain constructedwith Ch1TrcMEP and Ch1T7PsCKG, with very low downstream expression weachieved kaurene production up to 100 mg/L. This demonstrated that thePsCK enzyme can support high flux under balanced pathway expression. Inaddition, this study also provided insights about the complex non-linearbehavior on diterpene product profile under different pathway balance(FIGS. 5 and 6, Table 2). Such complex behavior on product selectivityof a pathway under varying flux modulations clearly demonstrates thepower of multivariate-modular pathway optimization. Under optimalbalance a strain can show high selectivity in product profile (FIG. 6D,strain 47). In addition, the multivariate-modular search allowedselection of the best variant kaurene enzyme (PsCK) to further engineerhyper-producing strains. When this optimal strain (i.e., Strain 47) wasgrown in a bioreactor system, we were able to—with minimal media orprocess improvements—generate a strain capable of 1.6 g/L production ofkaurene (FIG. 7). MMME also provides insight towards furtheroptimization of the pathway and helps identify the best variant of GGPPSenzyme (Table 1) using a similar approach. Furthermore, as observed inpathway engineering on taxadiene-producing strains, kaurene productionalso inversely correlated to the production of the inhibitory moleculeindole (FIG. 8).

(B) Engineering Steviol Biosynthesis in E. coli

The biosynthesis of steviol involves two key oxidation reactionsmediated by cytochrome P450 enzymes (FIG. 3). P450s are importantoxidizing enzymes involved in the metabolic pathways of thousands ofnatural products (25). Until recently, the scientific community believedthat when compared to native eukaryotic hosts (e.g. plants or yeast),bacterial hosts, such as E. coli, were not an ideal system forperforming this important natural product chemistry. However, whileoptimizing taxol biochemistry in E. coli, an understanding was developedof the mechanistic structure-function relationships responsible for thebiochemistry of P450 enzymes, specifically related to their use in E.coli. Optimal engineering of N-terminal membrane region and constructionof optimal combinations of CYP450 and the co-factor P450 reductase (CPR)enzymes is key for functional expression. Several enzyme/pathwayoptimization techniques were developed for the functional expressionCYP450 enzymes and in vivo oxidation of complex natural terpenoidnatural products such as taxadiene, valencene, limonene or kaurene.

Steviol biosynthesis is mediated by two different CYP450 enzymes,kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH) with a CYP450reductase (CPR). Several candidate genes/enzymes were identified andannotated as P450 enzymes for oxidation and hydroxylation reactions insteviol biosynthesis (Table 1). The functional expression of the enzymesKO and KAH for carboxylation and hydroxylation requires protein redesignand engineering. We started with redesigning and cloning the SrKO enzymefor improved functional expression in E. coli. After a thoroughbioinformatics analysis, several N-terminal truncated and modified KOenzymes were constructed (FIG. 9A). Constructs were created thatincorporate SrKO and co-factor cytrochrome P450 reductase enzyme (SrCPR)as a fusion protein (“linker” constructs) or as a polycistronic modules(“operon” constructs) (FIG. 9B) in the pET45d expression vector. Theproduction and relative solubility of the protein in these constructs inE. coli was assessed using SDS-PAGE analysis (FIG. 9C).

These constructs were then transferred into our production vector p5Trcto test the in vivo functional activity of the pathway. These constructswere transformed into kaurene producing strains 3, 9 and 11 (Table 2) totest the conversion of kaurene to kaurenoic acid (Table 3). The designedchimeric enzymes were functionally active, but the incomplete reactivityof the enzymes resulted in the production of kaurenol and kaurenal.Among all various N-terminal truncated KO constructs, the 39AAtruncation of KO was more functionally active compared to 4 and 20 aminoacid truncated constructs. Additionally, the SrKO and SrCPR expressed asoperons showed similar activity as fusion enzyme constructed from SrKOand SrCPR. Subsequent to this initial work, we further optimized theSrKO enzyme as part of a three-gene KAH-KO-CPR module, see below, and inthis construct the optimal SrKO construct has 20 amino acid residuestruncated from the N-terminus, resulting in complete conversion ofkaurene to kaurenoic acid (see below).

In the initial screening of the above SrKO strains, it was found thatthe availability of substrate pool for subsequent conversion tokaurenoic acid is important. When a high substrate (kaurene) pool wasavailable (strain 9) the oxidation pathway converted kaurene to ˜60 mg/Lof oxygenated kaurene compounds. Previous in vitro studies on theenzymatic activity of the Arabidopsis thaliana KO enzyme demonstratedthat the enzyme produces the alcohol, diol, and aldehyde derivatives ofkaurene (12). Similar product diversity is seen with taxol P450 enzymes,however, in that work rebalancing of the entire pathway changed theproduct profile and produced the hydroxylated taxanes exclusively.Therefore, strain engineering studies were initiated to rebalance the KOP450 module and upstream modules. In order to rebalance the modules, theKO pathway was transferred into the high kaurene-producingchromosomally-integrated strain (Strain 47) and tested for activity andproductivity (FIG. 10). Orthologous KO enzymes from different organismswere also designed, synthesized and tested (Table 1) to identify thebest variant enzymes. Multivariate pathway optimization was performed,as with the kaurene pathway, to identify the best variant enzyme andtheir non-linear product profiles and product distributions undervarying flux balances (FIG. 11). SrKO activity was subsequently improvedby designing and testing a collection of point mutants in the wild-typebackground (Table 4).

Upon successful production of kaurenoic acid, the final enzymatic stepin the biosynthetic pathway was incorporated and tested, hydroxylationat the C13 carbon of kaurenoic acid by the enzyme KAH to yield steviol(FIGS. 1 and 2). Studies on the polycistronic expression of SrKO andSrCPR proved that this enzymes can be expressed as independentcomponents and remain functionally active. It was determined whetherboth KO and KAH could be functionally active with a single SrCPR enzyme.In order to limit the number of plasmids and balance the expression ofKO and KAH, a single copy SrCPR was chromosomally integrated into akaurene engineered strain. The KO and KAH was constructed aspolycistronic expression under p5Trc plasmid. Detectable levels ofsteviol were detected in GC-MS analysis of all strains (Table 5) afterfour days fermentation and extraction with ethyl acetate.

With this promising initial result in hand, the optimal enzymes forassembly into a biosynthetic pathway in E. coli were identified by invivo expression of different engineered versions of both a KO and KAHcandidate in a polycistronic operon with a CPR co-factor enzyme (FIG.12). The AtKAH enzyme was further enhanced with a campaign of pointmutations. A rational approach was used to design a collection of singlepoint mutations in the AtKAH sequence, aimed at increasing stability,solubility, or activity of the wild-type enzyme for improved conversionof kaurenoic acid to steviol. The point mutations and correspondingfold-change improvements over wild-type AtKAH are summarized in Table 6,and are visualized in FIG. 13. Some of these point mutations were thenrecombined in the AtKAH enzyme, and a recombinant enzyme was identifiedthat was capable of complete conversion of kaurenoic acid to steviol(FIG. 14). When expressed in an operon with optimal SrKO and SrCPR,complete conversion of kaurene to steviol was demonstrated (FIG. 15),further highlighting the importance of careful balancing of pathwaycomponents.

(C) Engineering Small Molecule Glycosylation in E. coli

To support multiple glycosylation of steviol to rebaudioside M and allintermediate steviol glycosides, a series of modifications wereintroduced to the background E. coli strain intended to increase theamount of UDP-glucose available. A series of gene knock-outs and geneinsertions were made aimed at increasing carbon flux to UDP-glucose anddecreasing flux in pathways away from UDP-glucose not in keeping withsmall molecule glycosylation (i.e., glycogen synthesis and carbonstorage). The design enables the import of sucrose into the cell and itssplitting into fructose and glucose via the activity of sucrosephosphorylase. A subsequent series of knock-outs have altered primarymetabolism so as to force biomass to be synthesized using only fructoseas carbon source, leaving glucose to be funneled exclusively towardsUDP-glucose biosynthesis when the cells are grown using sucrose as acarbon source. However, the cells are still capable of growth andimproved UDP-glucose availability when grown on either glycerol orglucose as the carbon source. The specific modifications applied to theE. coli strain to enact this strategy are listed in Table 7. Thesemodifications were tested to determine whether they enabled enhancedglycosylation of small molecules, and demonstrated that they indeed doby showing enhanced in vivo glycosylation of caffeic acid (supplementedin the media) by the engineered strains (FIG. 16).

Having constructed the steviol core molecule, the core was glycosylatedwith UDP-glucose using an assembly of four UGTs, each capable ofdifferent glycosylation chemistries (Tables 8 and 9). These chemistriesinclude (1) O-glycosylation at C13 of steviol, (2) O-glycosylation atC19 of steviol, (3) 1-2′-glycosylation at either the C13 or C19O-glucose, and (4) 1-3′-glycosylation at either the C13 or C19 O-glucose(see FIG. 1 for an example incorporating all the above chemistries).Once one or both of the C13 and/or C19 oxygens are glycosylated, furtherglycosylations can be added to the O-glucose via 1-2′ or 1-3′ additions.As described below, these UGTs have been fundamentally modified by theshuffling of their domains, and further enhanced by point mutationsaimed and enhancing flux through to the desired end product of RebM. TheMMME approach was applied to rapidly combine the four UGTs in allpossible combinations and screen the resulting constructs in vivo in asteviol-producing strain background (FIG. 17). The improved UGTsassembled in the optimum polycistronic configuration were combined withthe four other modules in a single E. coli strain. We cultured thestrain and demonstrated in vivo production of steviol glycosides leadingto rebaudioside M (FIG. 18). The strain is capable of producing 5.7 mg/Lof total steviol glycosides, which includes 100 μg/L of RebM. Bygenerating all possible constructs and expressing them either inplasmids or integrated into the chromosome, under a variety of promoterstrengths, steviol glycoside product profiles were obtained withRebD:RebM ratios ranging from 1:1 all the way to 0:1 (no RebDremaining).

To our knowledge, this is the first time two cytochrome P450monooxygenases have been functionally expressed in E. coli for theproduction of a bifuncational oxygenated terpenoid molecule such assteviol. Additionally, a single CPR enzyme acted as co-factor for bothP450 enzymes (KO and KAH) for converting kaurene to steviol. This isanother significant leap in the engineering of P450 mediated oxidationchemistry in E. coli system. Moreover, to our knowledge, this is thefirst time four UGTs have been combined in a single E. coli strain anddemonstrated to be capable of performing six sequential glycosylationsof a terpenoid core molecule to produce rebaudioside M, let alone theintermediate steviol glycosides. This is a significant leap forward inthe engineering of UGTs and the establishment of a platform forsustainable production of rare steviol glycosides.

Example 2: Construction of Circular Permutants of GlycosyltransferaseEnzymes

Natural selection acting on an enzyme tends to select for sufficientstability and activity for the biological function. This process sendsan enzyme down a specific evolutionary path that may make it not readilycompatible with the stability and activity gains needed for industrialapplications. As an example, enzymes specialized for a specificsubstrate tend to be more challenging to engineer for new substratesthan enzymes that have not been specialized. Thus, ‘shaking up’ anenzyme by swapping domain connections might create an enzyme with thesame protein fold, yet with novel folding and folded interactions thatwould make it newly-amenable to selection and evolution. In other words,we might be able to ‘jump’ a protein fold to another point inevolutionary space simply by shuffling the sequence, moving the enzymeaway from its original evolutionary path without introducing any aminoacid mutations.

UGTs (UDP-glucose glycosyltransferases) have two domains, a morevariable N-terminal substrate binding (sugar acceptor) domain and a moreconserved C-terminal UDP-glucose binding (sugar donor) domain. TheN-terminal domain is mostly determinant of substrate specificity for theenzyme, but some specificity is controlled by the C-terminal domain.Each of these domains makes up roughly half of the protein. Given thistwo-domain structure, we hypothesized that cutting the protein in halfto create new N- and C-termini and attaching the originals together(e.g., circular ‘permutization’) would ‘shuffle’ the enzyme and createnew opportunities for engineering improved activity (since the resultingenzyme would not be the result of selective pressure).

As a description of general procedure, designing a shuffled enzymeinvolves the following steps: (i) create a homology model to a known UGTwith desired glycosylation activity (FIG. 19); (ii) using the homologymodel, estimate distance between N- and C-terminal residues; (iii)design linkers of various lengths to connect the existing N- andC-termini; (iv) select positions in the enzyme to become the new N- andC-termini; (v) synthesize the resulting sequences; (vi) express in vivoand in vitro and identify any shuffled enzymes that retain parentactivity; (vii) modify designs via rational engineering, informed by anew homology model of the shuffled enzyme; and (viii) repeat step viiuntil desired activity improvement is achieved. When creating linkers(step iii), identify the N- and C-termini residues closest to ends, butpredicted to be directly interacting with the rest of the protein basedon the structure in the homology model (FIG. 20). When choosing cutsites for new N- and C-termini (step iv) (FIG. 21), choose a loop regionbetween secondary structure elements, which maintains domain structure,is close to the middle of the sequence as possible, and which is solventexposed and away from the active site.

Circular permutants were designed, synthesized, and incorporated foreach of the C13, C19, 1-2′ and 1-3′ glycosylating activities (parentenzymes are SrUGT85C2, SrUGT74G1, OsUGT1-2 (Q0DPB7_ORYSJ) (28), andSrUGT76G1, respectively). All four activities resulted in workingcircular permutants. As an example, data demonstrating the first roundof circular permutant engineering resulting in the novel enzymeMbUGT1-2, showing equivalent activity with the parent enzyme OsUGT1-2(FIG. 22). A subsequent round of refinement to the shuffled enzyme hasgenerated enzymes with enhanced activities compared to the originalparent sequence, demonstrating the potential for this shuffling approachto generate improved enzymes (FIG. 23). These refinements focus onfiner-scale modifications to the position and specific residues formingthe novel N and C-termini of the shuffled enzyme, as well as refiningthe length and amino acid sequence of the linker connecting the parentalN and C-termini.

A series of point mutations to the MbUGT1-2 enzyme has resulted infurther improvement to this novel sequence (FIGS. 24 and 25), confirmingthat the process has created opportunities for significant improvementby shuffling the enzyme sequence. However, when these point mutationsconferring improved activity in MbUGT1-2 were tested in the parentOsUGT1-2 background, they were found to be either neutral or deleteriousto activity (FIG. 26). The lack of transferability of point mutationsbetween the novel enzyme and the parent sequence confirms that circularpermutization is a valuable and general approach for creatingopportunities for UGT enzyme improvement.

Example 3: Chimeric Fusions of Distinct Glycosyltransferases

In a similar vein to the shuffling of enzymes engendered by circularpermutization, an alternate method to shuffle glycosyltransferaseenzymes was created by swapping the N-terminal small molecule sugaracceptor binding domain or the C-terminal sugar-donor binding domainbetween known UGTs. For example, a chimeric enzyme composed of theN-terminal domain of SrUGT85C2 and the C-terminal domain of SrUGT76G1was created (FIG. 27). The rationale behind this approach rests on theconcept of shuffling the domains of a UGT enzyme, only this time we addthe nuance of shuffling domains between UGTs. The intent is to generatea non-optimized enzyme with a novel sequence, capable of furtherevolution away from the point in the energy landscape occupied by theparent enzyme, and towards a new optimum enzyme configuration in theproduction strain. Again, given that this enzyme is not a result ofnatural selection per se, the shuffled enzyme resulting from thischimeric approach should have increased evolutionary potential/greaterpotential to benefit positively from point mutations (i.e., withincreased activity). Moreover, this approach can be used to generatechimeric protein with enhanced folding and/or stability.

In brief, this approach employs four broad steps: (1) identify twocandidate UGTs; (2) select crossover positions for making a chimerabetween the two UGTs (i.e., select the point at which to join the twosequences); (3) mutate the C-terminal domain (thenucleotide-carbohydrate binding domain, e.g. UDP-glucose binding domain)to improve interaction with the small molecule substrate or theN-terminal small-molecule binding domain, based on structuralconsiderations; (4) create and test chimeric constructs for activity.This approach is generalizable and applicable for improving thefunctional performance of potentially any UGT. Given the conserveddomain structure of UGTs, domains from any two UGTs could be recombined.

Example 4: Modifying Glycosyltransferase Enzymes for Improved Activityand Biosynthesis of Rare Glycosides

Although only around 20 steviol glycosides occur in sufficientquantities to have been characterized from stevia plants, there areseveral more possible steviol glycosides with different glycosylationpatterns that can be created biosynthetically. Table 10 and FIGS. 29,30, 31 and 32 summarize the known and potential steviol glycosidesdescribed in this section, which are abbreviated with the symbol SG#.Some of these glycosides exist in nature, and others arebiosynthetically possible using UGTs that catalyze the fourglycosylation chemistries described herein (i.e., C13-O-glycosylation,C19-O-glycosylation, 1,2′-glycosylation, and 1,3′-glycosylation).

A rational design approach was used to design a collection of single,double, and triple point mutations in the SrUGT85C2 sequence (possessingC13-O-glycosylating activity), aimed at increasing stability,solubility, or activity of the wild-type enzyme for improved conversionof steviol to steviolmonoside (SG1), and/or C19-glucopyranosyl steviol(SG2) to rubusoside (SG5), and/or SG4 to SG10, and/or SG7 to SG11,and/or SG13 to SG17, and/or SG19 to SG29, and/or SG23 to SG31. The pointmutations and corresponding fold-change improvements over wild-typeSrUGT85C2 are summarized in Table 11, and are visualized in FIGS. 32 and33.

A rational design approach was used to design a collection of single,double, and triple point mutations in the SrUGT74G1 sequence (possessingC19-O-glycosylating activity), aimed at increasing stability,solubility, or activity of the wild-type enzyme for improved conversionof steviol to C19-glucopyranosyl steviol (SG2), and/or steviolmonoside(SG1) to rubusoside (SG5), and/or steviolbioside (SG3) to stevioside(SG8), and/or SG6 to rebaudioside G (SG9), and/or rebaudioside B (SG12)to rebaudioside A (SG16), and/or SG18 to SG28, and/or SG22 to SG30.

A rational design approach was used to design a collection of single,double, and triple point mutations in the MbUGT1-2 sequence (possessing1-2′ glycosylating activity), aimed at increasing stability, solubility,or activity of the wild-type enzyme for improved conversion ofsteviolmonoside (SG1) to steviolbioside (SG3), and/or C19-glucopyranosylsteviol (SG2) to SG4, and/or rubusoside (SG5) to stevioside (SG8),and/or rubusoside (SG5) to SG10, and/or rubusoside (SG5) to rebaudiosideE (SG14), and/or SG6 to rebaudioside B (SG12), and/or SG7 to SG13,and/or stevioside (SG8) to rebaudioside E (SG14), and/or SG10 torebaudioside E (SG14), and/or rebaudioside G (SG9) to rebaudioside A(SG16), and/or rebaudioside G (SG9) to SG21, and/or SG11 to SG17, and/orSG11 to SG20, and/or rebaudioside B (SG12) to SG22, and/or SG13 to SG23,and/or SG15 to rebaudioside I (SG26), and/or SG15 to SG27, and/orrebaudioside A (SG16) to rebaudioside D (SG24), and/or rebaudioside A(SG16) to SG30, and/or SG17 to SG25, and/or SG17 to SG31, and/or SG20 toSG25, and/or SG21 to rebaudioside D (SG24), and/or rebaudioside D (SG24)to SG37, and/or SG25 to SG39, and/or rebaudioside I (SG26) torebaudioside M (SG32), and/or rebaudioside I (SG26) to SG38, and/or SG27to rebaudioside M (SG32), and/or SG27 to SG40, and/or SG28 to SG33,and/or SG29 to SG35, and/or SG30 to SG37, and/or SG31 to SG39, and/orrebaudioside M (SG32) to SG43, and/or rebaudioside M (SG32) to SG44,and/or SG34 to SG41, and/or SG36 to SG42, and/or SG38 to SG43, and/orSG40 to SG44, and/or SG41 to SG47, and/or SG42 to SG48, and/or SG43 toSG46, and/or SG44 to SG46. The point mutations and correspondingfold-change improvements over wild-type MbUGT1-2 are summarized in Table12, and representative reactions are shown in FIGS. 24 and 25.

A rational design approach was used to design a collection of single,double, triple, or quadruple point mutations in the SrUGT76G1 sequence(possessing 1-3′ glycosylating activity), aimed at increasing stability,solubility, or activity of the wild-type enzyme for improved conversionof steviolmonoside (SG1) to SG6, and/or C19-glucopyranosyl steviol (SG2)to SG7, and/or steviolbioside (SG3) to rebaudioside B (SG12), and/or SG4to SG13, and/or rubusoside (SG5) to rebaudioside G (SG9), and/orrubusoside (SG5) to SG11, and/or rubusoside (SG5) to SG15, and/orstevioside (SG8) to rebaudioside A (SG16), and/or stevioside (SG8) toSG20, and/or rebaudioside G (SG9) to SG15, and/or SG10 to SG17, and/orSG10 to SG21, and/or SG11 to SG15, and/or rebaudioside B (SG12) to SG18,and/or SG13 to SG19, and/or rebaudioside E (SG14) to rebaudioside D(SG24), and/or rebaudioside E (SG14) to SG25, and/or rebaudioside E(SG14) to rebaudioside M (SG32), and/or rebaudioside A (SG16) torebaudioside I (SG26), and/or rebaudioside A (SG16) to SG28, and/or SG17to SG27, and/or SG17 to SG29, and/or SG20 to rebaudioside I (SG26),and/or SG21 to SG27, and/or rebaudioside D (SG24) to rebaudioside M(SG32), and/or rebaudioside D (SG24) to SG33, and/or SG25 torebaudioside M (SG32), and/or SG25 to SG35, and/or rebaudioside I (SG26)to SG34, and/or SG27 to SG36, and/or SG28 to SG34, and/or SG29 to SG36,and/or SG30 to SG38, and/or SG31 to SG40, and/or rebaudioside M (SG32)to SG41, and/or rebaudioside M (SG32) to SG42, and/or SG33 to SG41,and/or SG35 to SG42, and/or SG37 to SG43, and/or SG39 to SG44, and/orSG41 to SG45, and/or SG42 to SG45, and/or SG43 to SG48, and/or SG44 toSG47. The point mutations and corresponding fold-change improvementsover wild-type SrUGT76G1 are summarized in Table 13, and representativereactions are shown in FIGS. 35 and 36.

Example 5: Improving Yield and Performance Above 22° C.

The performances of the enzymes in the kaurene module were determined tobe suboptimal at temperatures above 22° C. A cluster of alternativeenzymes were identified for the GGPPS (geranylgeranyl diphosphate)synthase enzyme and the bi-functional copalyl diphosphate (CPP)/kaurenesynthase enzymes used in the previous examples. In particular, alternateenzymes from bacterial sources were considered, reasoning that these mayfunction better in E. coli than plant and fungal enzymes. Enzymes fromthermophilic bacteria were considered where possible. For the CPPsynthase and kaurene synthase activities, genes from bacteria in therhizosphere were identified, since they are often kaurene-producing dueto their symbiotic lifestyle.

FIG. 39 shows the results for alternate GGPPS enzymes. Several enzymesshow improved performance at higher temperatures, including Marinebacterium 443, Synechoccus sp., Thermotoga maritima, Cornybacteriumglutamicum, and Pyrobaculum calidifontis.

FIG. 40 shows the results for alternate CPPS and KS enzymes. Erwinatracheiphila (Et)CPPS and EtKS showed improved activity at highertemperatures.

Production of various steviol glycosides (including Reb M) was tested at22° C. in a select strain. The strain was E. coli K12 with a pBACsingle-copy chromosome containing FAB46-MEP, T7-PsCKS-AnGGPPS,T7-AtKAH-SrKO-SrCPR, T7-MbUGT1,3-MbUGT1,2-MbUGTc13-MbUGTc19. As shown inFIG. 41, Reb M titer was 55.5 mg/L with a total steviol glycoside titerof 58.3 mg/L, which is equal to 94.4% Reb M. The Reb M:Reb D ratio was64.5:1 (in grams).

Statistic Quantity Titer, Total Steviol Glycosides (mg/L) 58.3 mg/LTiter, Rebaudioside M (mg/L) 55.5 mg/L % Reb M (of total glycosides)94.4% Reb M: Reb D (g/g) 64.5:1

The intracellular accumulation of steviol glycosides was investigated.As shown in FIG. 42, the majority of the steviol glycosides are excretedfrom the cell. FIG. 42 shows the combined intracellular andextracellular material, as a percentage of product accumulating insidethe cell versus outside. This was in contrast to initial studies havingsubstantially less yield of steviol glycosides, which saw mostlyintracellular accumulation. It s possible that the initial studies wereof such low titer that accumulated product pools were insufficient forthe active transport mechanisms required to pump the product out of thecells. Indeed, as the titer increased, a greater proportion of theproduct accumulated outside the cell, indicating that once above thethreshold concentration for the putative pump activity, the rest of theproducts get moved out. These data are very promising from a strainengineering perspective and commercial production in E. coli, since ifintermediate product pools are maintained below the Kb of thetransporter, we can effectively push C-flux through to the end productwithout losing carbon to the outside (e.g., once a steviol glycosideintermediate is pumped out, it can no longer be further glycosylated tothe desired product, such as RebM).

Point mutants of UGT85C2 were generated, and tested at 22, 30, and 34°C. FIG. 43A, B show steviol monoside production at 34° C. FIG. 43C showsproduction of steviolmonoside with selected mutants at 22, 30, and 34°C. Several mutations showed higher production of steviolmonoside at 34°C., with P215T being the highest producing mutation.

Circular permutants of 74G1 were also tested for activity at 30 and 34°C. FIG. 44A,B show conversion of steviol to 13C-c19-Glu-Steviol (FIG.44A) and steviolbioside to 13C Stevioside (FIG. 44B).

Mutations in AtKAH were screened for activity at 22, 26, and 30° C.C331I provided substantial thermostability, as shown in FIG. 45. C331Iwas made in the t14 background.

MbUGT1,2 rational recombinations were made, and screened at 30 and 34°C. for conversion of Reb A to Reb D (FIG. 46A), as well as forconversion of Steviolmonoside to 13c Steviolbioside (FIG. 46B). Thesestudies resulted in a circular permutant truncated to create a newN-terminus at residue 196, with mutations introduced at S16W, H422E,R430E, R434H (MbUGT1,2-2). In these studies, cells producing theseenzymes were induced for 4 hours of protein production at the listedtemperature, extracted, and assayed in vitro overnight. Substrateconcentration is 1 mM.

The effect of temperature on kaurene substrate production at 30 and 34°C. was tested. FIG. 47 shows kaurene production at 30° C. across variousmodule constructs and FIG. 48 shows kaurene production at 34° C. acrossvarious module constructs. At 30° C., Ch1.T7-PsCK-AnGGPPS in T7MEPbackground gave highest kaurene titers (˜15 mg/L). At 34° C.,Ch1.T7-PsCK-AnGGPPS in T7MEP background showed the highest kaurenetiters (˜2 mg/L).

To investigate the thermotolerance of AtKAH, AtKAH point mutants weretested at 30° C. and 34° C. Conditions were: R media+glucose, 96 deepwell plate, 3 days at 30° C. or 34° C. The strain background wasp5Trc-(8RP)t14AtKAH-O-(8RP)t20SrKO-O-FLSrCPR. FIG. 49 shows productionof Steviol at 30° C. across a library of AtKAH point mutations. FIG. 50shows production of Steviol at 34° C. across a library of AtKAH pointmutations. Various point mutations show improved thermotolerance thatwild type, as shown by higher titers of steviol at 30 or 34° C.

MbUGT1_2 curcular permutants were tested for activity at 30, 33, and 37°C. FIG. 51(A),(B) shows activities of MbUGT1_2 circular permutants at30° C., 34° C., and 37° C. Panel (A) shows conversion of Reb A to Reb D,while Panel (B) shows conversion of Steviolmonoside to 13CSteviolbioside. For both, expression of circular permutants was induced,followed by a four hour incubation period. As shown, EUGT11 lost itsactivity when induced at and above 30° C. In contrast, lead circularpermutants seem to be most active at 30° C. MbUGT1_2 196L retainshighest activity on both substrates.

FIG. 52 shows activities of UGT85C2 mutants for conversion of Steviol to13C Steviolmonoside at 30° C., 34° C., and 37° C. Expression wasinduced, followed by a four hour incubation period. As shown, 85C2-WTand the leads retain comparable activity at 34° C. and 37° C.,maintaining highest activity at 30° C.

FIG. 53 shows activities of UGT76G1 mutants for conversion of Reb D to13C Reb M at 30° C., 34° C., and 37° C. Expression was induced, followedby a four hour incubation period. 76G1-L200A is particularly active wheninduced and assayed at the higher temperatures, possibly due to agreater amount of protein.

FIG. 54 shows activities of UGT74G1 circular permutants for conversionof Steviolbioside to 13C Stevioside at 30° C., 34° C., and 37° C.74G1-WT retains activity on Steviolbioside even when induced and assayedat 37 C. The circular permutants 74G1-259M and 74G1-259L show asignificant drop in activity at higher temperatures.

REFERENCES

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TABLE 1 Summary of enzyme/gene sequences enabling biosynthesis ofsteviol. No. Enzyme Species Gene ID Protein ID 1 TcGGPPS Taxuscanadensis AF081514.1 AAD16018.1 2 AgGGPPS Abies grandis AF425235.2AAL17614.2 3 AnGGPPS Aspergillus nidulans XM_654104.1 XP_659196.1 4SmGGPPS Streptomyces melanosporofaciens AB448947.1 BAI44337.1 5 MbGGPPSMarine bacterium 443 n/a AAR37858.1 6 PhGGPPS Paracoccus haeundaensisn/a AAY28422.1 7 CtGGPPS Chlorobium tepidum TLS NC_002932.3 NP_661160.18 SsGGPPS Synechococcus sp. JA-3-3Ab n/a ABC98596.1 9 Ss2GGPPSSynechocystis sp. PCC 6803 n/a BAA16690.1 10 TmGGPPS Thermotoga maritimaHB8 n/a NP_227976.1 11 CgGGPPS Corynebacterium glutamicum n/aNP_601376.2 12 TtGGPPS Thermus thermophillus HB27 n/a YP_143279.1 13PcGGPPS Pyrobaculum calidifontis JCM 11548 n/a WP_011848845.1 14 SrCPPSStevia rebaudiana AF034545.1 AAB87091.1 15 EtCPPS Erwina tracheiphilan/a WP_020322919.1 16 SfCPPS Sinorhizobium fredii n/a WP_010875301.1 17SrKS Stevia rebaudiana AF097311.1 AAD34295.1 18 EtKS Erwina tracheiphilan/a WP_020322918.1 19 SfKS Sinorhizobium fredii n/a WP_010875302.1 20GfCPPS/KS Gibberella fujikuroi AB013295.1 Q9UVY5.1 21 PpCPPS/KSPhyscomitrella patens AB302933.1 BAF61135.1 22 PsCPPS/KS Phaeosphaeriasp. L487 AB003395.1 O13284.1 23 AtKO Arabidopsis thaliana NM_122491.2NP_197962.1 24 SrKO Stevia rebaudiana AY364317.1 AAQ63464.1 25 PpKOPhyscomitrella patens AB618673.1 BAK19917.1 26 AtCPR Arabidopsisthaliana X66016.1 CAA46814.1 27 SrCPR Stevia rebaudiana DQ269454.4ABB88839.2 28 AtKAH Arabidopsis thaliana NM_122399.2 NP_197872.1 29SrKAH1 Stevia rebaudiana DQ398871.3 ABD60225.1 30 SrKAH2 Steviarebaudiana n/a n/a

TABLE 2 Strains constructed to evaluate pathways for kaurenebiosynthesis. Strain # Upstream Downstream 1 WT Ch1.T7-KCG 2 Ch1.Trc-MEPCh1.T7-KCG 3 Ch1.T7-MEP Ch1.T7-KCG 4 WT p5-Trc-KCG 5 Ch1.Trc-MEPp5-Trc-KCG 6 Ch1.T7-MEP p5-Trc-KCG 7 WT p10-Trc-KCG 8 Ch1.Trc-MEPp10-Trc-KCG 9 Ch1.T7-MEP p10-Trc-KCG 10 WT p20-Trc-KCG 11 Ch1.Trc-MEPp20-Trc-KCG 12 Ch1.T7-MEP p20-Trc-KCG 13 WT p5-T7-KCG 14 Ch1.Trc-MEPp5-T7-KCG 15 Ch1.T7-MEP p5-T7-KCG 16 WT Ch1.T7-PpCKG 17 Ch1.Trc-MEPCh1.T7-PpCKG 18 Ch1.T7-MEP Ch1.T7-PpCKG 19 WT p5-Trc-PpCKG 20Ch1.Trc-MEP p5-Trc-PpCKG 21 Ch1.T7-MEP p5-Trc-PpCKG 22 WT p10-Trc-PpCKG23 Ch1.Trc-MEP p10-Trc-PpCKG 24 Ch1.T7-MEP p10-Trc-PpCKG 25 WTp20-Trc-PpCKG 26 Ch1.Trc-MEP p20-Trc-PpCKG 27 Ch1.T7-MEP p20-Trc-PpCKG28 WT p5-T7-PpCKG 29 Ch1.Trc-MEP p5-T7-PpCKG 30 Ch1.T7-MEP p5-T7-PpCKG31 WT Ch1.T7-GfCKG 32 Ch1.Trc-MEP Ch1.T7-GfCKG 33 Ch1.T7-MEPCh1.T7-GfCKG 34 WT p5-Trc-GfCKG 35 Ch1.Trc-MEP p5-Trc-GfCKG 36Ch1.T7-MEP p5-Trc-GfCKG 37 WT p10-Trc-GfCKG 38 Ch1.Trc-MEP p10-Trc-GfCKG39 Ch1.T7-MEP p10-Trc-GfCKG 40 WT p20-Trc-GfCKG 41 Ch1.Trc-MEPp20-Trc-GfCKG 42 Ch1.T7-MEP p20-Trc-GfCKG 43 WT p5-T7-GfCKG 44Ch1.Trc-MEP p5-T7-GfCKG 45 Ch1.T7-MEP p5-T7-GfCKG 46 WT Ch1.T7-PsCKG 47Ch1.Trc-MEP Ch1.T7-PsCKG 48 Ch1.T7-MEP Ch1.T7-PsCKG 49 WT p5-Trc-PsCKG50 Ch1.Trc-MEP p5-Trc-PsCKG 51 Ch1.T7-MEP p5-Trc-PsCKG 52 WTp10-Trc-PsCKG 53 Ch1.Trc-MEP p10-Trc-PsCKG 54 Ch1.T7-MEP p10-Trc-PsCKG55 WT p20-Trc-PsCKG 56 Ch1.Trc-MEP p20-Trc-PsCKG 57 Ch1.T7-MEPp20-Trc-PsCKG 58 WT p5-T7-PsCKG 59 Ch1.Trc-MEP p5-T7-PsCKG 60 Ch1.T7-MEPp5-T7-PsCKG

TABLE 3 Combinations of upstream and downstream pathway configurationstested for KO ctivity and kaurenoic acid biosynthesis. Ch1 = 1 copychromosomally integrated, p5/p10/p20 = plasmids of increasing copynumber, Trc/T7 = promoters of increasing ranscriptional strength.Ch1.T7-MEP Ch1.T7-MEP Ch1.T7-MEP Upstream/Downstream Ch1.T7-SrKCGp10Trc-SrKCG p20Trc-SrKCG p5Trc-(8RP)t4SrKO-

L-t69SrCPR p5Trc-(8RP)t20SrKO-

L-t69SrCPR p5Trc-(8RP)t39SrKO-

L-t69SrCPR p5Trc-(8RP)t39SrKO-

(8RP)t69SrCPR p5Trc-(MA)t39SrKO-

(8RP)t69SrCPR

TABLE 4 Fold-change in in vivo activity over parental enzyme for pointmutants of SrKO. The fold increases describe the change in kaureneremaining in this strain, or the change in kaurenoic acid produced, bothrelative to the wild-type (non-mutated) enzyme-bearing parental strain.Wild-type Fold increase Fold increase in residue Position Mutation inkaurene kaurenoic acid A 116 R 1.0 1.8 T 119 S 0.9 1.9 I 183 V 1.0 1.7 H382 Y 1.0 1.8

TABLE 5 Combinations of upstream and downstream pathway configurationstested for KO/KAH activity and steviol biosynthesis. Ch1 = 1 copychromosomally integrated, p5 and p10 = plasmids of increasing copynumber, Trc/T7 = promoters of increasing transcriptional strength.Steviol Expression module Detected Ch1.T7-MEPp5Trc-(8RP)t39SrKO-(8RP)t7SrKAH ++ Ch1.T7-p5Trc-(8RP)t39SrKO-(8RP)t21SrKAH ++ (8RP)t69SrCPRp5Trc-(8RP)t39SrKO-(8RP)t29SrKAH + p10Trc-SrKCG

TABLE 6 Fold-change in activity over parental enzyme for point mutantsof AtKAH. The fold increases describe the change in kaurenoic acidremaining in this strain, or the change in steviol produced, bothrelative to the wild-type (non-mutated) enzyme-bearing parental strain.Wild-type Wild-type Fold increase Fold increase residue PositionMutation residue Position Mutation kaurenoic acid steviol A 25 L 0.7 1.6K 37 R 0.8 1.4 S 79 T 0.7 1.6 F 84 I 1.3 0.0 F 84 M 1.2 0.2 Y 95 F 1.11.0 H 104 I 1.4 0.0 I 107 M 1.0 1.2 L 116 M 1.4 0.9 T 119 C 0.7 1.5 N123 D 0.9 1.1 R 126 K 1.4 0.0 I 127 P 1.1 0.0 I 127 V 1.1 0.1 I 130 L1.0 0.0 L 134 V 1.3 0.7 I 137 L 0.3 1.9 I 142 L 1.2 0.8 I 142 V 0.5 2.1I 143 L 1.7 0.0 R 155 K 0.9 2.0 T 162 F 1.4 0.0 H 163 M 1.2 0.2 I 166 V0.3 1.2 M 180 L 1.3 1.5 V 188 I 1.0 1.1 E 193 G 1.1 1.7 C 196 A 0.9 1.5D 197 E 1.7 0.8 V 207 F 1.4 0.5 A 213 S 0.7 0.9 C 216 A C 325 V 0.8 0.9C 216 I 1.1 1.0 C 216 S 1.3 0.0 A 226 E 0.9 1.5 I 231 L 0.3 0.8 L 235 Q0.3 2.4 I 238 M 1.5 0.2 L 244 F 0.9 1.4 F 245 L 1.6 0.0 F 245 V 1.5 0.0R 246 S 1.1 0.8 F 247 L 1.0 1.1 L 272 I 1.7 0.0 S 274 D 1.0 1.3 S 275 L0.6 1.2 I 285 R 0.6 1.7 C 287 S 0.7 1.7 K 292 E 1.2 0.6 Q 297 E 1.0 0.9C 307 S 0.6 0.0 V 322 I 1.1 1.4 C 325 I 0.5 2.4 C 325 M 0.6 2.2 F 330 L1.0 1.5 D 334 E 0.3 2.2 S 335 T 0.7 0.5 S 339 T 0.2 2.2 N 350 H 0.9 1.1S 352 E 0.7 1.5 S 363 E 0.8 1.3 E 373 D 1.1 1.6 I 375 L 1.2 0.8 V 381 L1.3 0.7 M 389 L 1.2 0.9 I 397 F 0.8 1.7 C 418 N 1.4 0.5 S 446 A 0.6 1.2E 447 N 0.8 1.4 C 453 P 0.7 0.0 I 460 M 1.3 0.2 V 470 L 0.7 1.7 G 475 A1.1 1.3 M 477 V 0.8 0.6 V 487 L 1.3 1.0 T 493 S 1.1 1.2 T 497 N 0.5 1.0Q 499 V 0.6 1.6 S 503 A 0.3 1.0 H 504 F 1.1 0.2 K 505 R 0.5 0.9 L 506 M1.6 0.0 L 507 I 1.5 0.0 L 507 T 1.6 0.0 L 507 V 1.5 0.0

TABLE 7 Modifications to E. coli strain to improve UDP-glucose substratepools and support high-titer production of steviol glycosides.Modification Type Gene ID (BioCyc) ΔgalE Deletion EG10362 ΔgalT DeletionEG10366 ΔgalK Deletion EG10363 ΔgalM Deletion EG11698 ΔushA DeletionEG11060 Δagp Deletion EG10033 Δpgm Deletion EG12144 galU (Escherichiacoli K-12 Insertion EG11319 substr. MG1655) ugpA (BifidobacteriumInsertion BBPR_0976 bifidum PRL2010) spl (Bifidobacterium adolescentisInsertion BAD_0078 ATCC 15703)

TABLE 8 Enzymes known to catalyze reactions required for steviolglycoside biosynthesis [to RebM]. Type of Substrate Productglycosylation Enzyme 1 Enzyme 2 Enzyme 3 Enzyme 4 SteviolSteviolmonoside C13 SrUGT85C2 Steviol C19-Glu-Steviol C19 SrUGT74G1MbUGTc19 Steviolmonoside Steviolbioside 1-2′ SrUGT91D1 SrUGT91D2OsUGT1-2 MbUGT1-2 Steviolmonoside Rubusoside C19 SrUGT74G1 MbUGTc19C19-Glu-Steviol Rubusoside C13 SrUGT85C2 Steviolbioside Stevioside C19SrUGT74G1 MbUGTc19 Steviolbioside RebB 1-3′ SrUGT76G1 Stevioside RebE1-2′ SrUGT91D1 SrUGT91D2 OsUGT1-2 MbUGT1-2 Stevioside RebA 1-3′SrUGT76G1 RebB RebA C19 SrUGT74G1 MbUGTc19 RebE RebD 1-3′ SrUGT76G1 RebARebD 1-2′ SrUGT91D1 SrUGT91D2 OsUGT1-2 MbUGT1-2 RebD RebM 1-3′ SrUGT76G1

TABLE 9 Summary of enzyme/gene sequences for biosynthesis of steviolglycosides, including RebM. Type of glycosylation Enzyme Gene ID ProteinID Description C13 SrUGT85C2 AY345978.1 AAR06916.1 C19 SrUGT74G1AY345982.1 AAR06920.1 MbUGTc19 — — circular permutant of SrUGT74G1 1-2′SrUGT91D1 AY345980.1 AAR06918.1 SrUGT91D2 ACE87855.1 ACE87855.1SrUGT91D2e — — US2011/038967 OsUGT1-2 NM_001057542.1 NP_001051007.2 WO2013/022989 MbUGT1-2 — — circular permutant of OsUGT1-2 1-3′ SrUGT76G1FB917645.1 CAX02464.1

TABLE 10 Summary of steviol glycoside structures.

Symbol Common Name R1 R2 Glycosylations SG1 Steviolmonoside Glcβ1- H— 1SG2 C19-glucopyranosyl H— Glcβ1- 1 steviol SG3 SteviolbiosideGlcβ1-2Glcβ1- H— 2 SG4 — H— Glcβ1-2Glcβ1- 2 SG5 Rubusoside Glcβ1- Glcβ1-2 SG6 — Glcβ1-3Glcβ1- H— 2 SG7 — H— Glcβ1-3Glcβ1- 2 SG8 SteviosideGlcβ1-2Glcβ1- Glcβ1- 3 SG9 Rebaudioside G Glcβ1-3Glcβ1- Glcβ1- 3 SG10 —Glcβ1- Glcβ1-2Glcβ1- 3 SG11 — Glcβ1- Glcβ1-3Glcβ1- 3 SG12 Rebaudioside BGlcβ1-2(Glcβ1-3)Glcβ1- H— 3 SG13 — H— Glcβ1-2(Glcβ1-3)Glcβ1- 3 SG14Rebaudioside E Glcβ1-2Glcβ1- Glcβ1-2Glcβ1- 4 SG15 — Glcβ1-3Glcβ1-Glcβ1-3Glcβ1- 4 SG16 Rebaudioside A Glcβ1-2(Glcβ1-3)Glcβ1- Glcβ1- 4 SG17— Glcβ1- Glcβ1-2(Glcβ1-3)Glcβ1- 4 SG18 — Glcβ1-3Glcβ1-2(Glcβ1- H— 43)Glcβ1- SG19 — H— Glcβ1-3Glcβ1-2(Glcβ1- 4 3)Glcβ1- SG20 — Glcβ1-2Glcβ1-Glcβ1-3Glcβ1- 4 SG21 — Glcβ1-3Glcβ1- Glcβ1-2Glcβ1- 4 SG22 —Glcβ1-2(Glcβ1-2Glcβ1- H— 4 3)Glcβ1- SG23 — H— Glcβ1-2(Glcb1-2Glcβ1- 43)Glcβ1- SG24 Rebaudioside D Glcβ1-2(Glcβ1-3)Glcβ1- Glcβ1-2Glcβ1- 5 SG25— Glcβ1-2Glcβ1- Glcβ1-2(Glcβ1-3)Glcβ1- 5 SG26 Rebaudioside IGlcβ1-2(Glcβ1-3)Glcβ1- Glcβ1-3Glcβ1- 5 SG27 — Glcβ1-3Glcβ1-Glcβ1-2(Glcβ1-3)Glcβ1- 5 SG28 — Glcβ1-3Glcβ1-2(Glcβ1- Glcβ1- 5 3)Glcβ1-SG29 — Glcβ1- Glcβ1-3Glcβ1-2(Glcβ1- 5 3)Glcβ1- SG30 —Glcβ1-2(Glcb1-2Glcβ1- Glcβ1- 5 3)Glcβ1- SG31 — Glcβ1-Glcβ1-2(Glcb1-2Glcβ1- 5 3)Glcβ1- SG32 Rebaudioside MGlcβ1-2(Glcβ1-3)Glcβ1- Glcβ1-2(Glcβ1-3)Glcβ1- 6 SG33 —Glcβ1-3Glcβ1-2(Glcβ1- Glcβ1-2Glcβ1- 6 3)Glcβ1- SG34 —Glcβ1-3Glcβ1-2(Glcβ1- Glcβ1-3Glcβ1- 6 3)Glcβ1- SG35 — Glcβ1-2Glcβ1-Glcβ1-3Glcβ1-2(Glcβ1- 6 3)Glcβ1- SG36 — Glcβ1-3Glcβ1-Glcβ1-3Glcβ1-2(Glcβ1- 6 3)Glcβ1- SG37 — Glcβ1-2(Glcb1-2Glcβ1-Glcβ1-2Glcβ1- 6 3)Glcβ1- SG38 — Glcβ1-2(Glcb1-2Glcβ1- Glcβ1-3Glcβ1- 63)Glcβ1- SG39 — Glcβ1-2Glcβ1- Glcβ1-2(Glcb1-2Glcβ1- 6 3)Glcβ1- SG40 —Glcβ1-3Glcβ1- Glcβ1-2(Glcb1-2Glcβ1- 6 3)Glcβ1- SG41 —Glcβ1-3Glcβ1-2(Glcβ1- Glcβ1-2(Glcβ1-3)Glcβ1- 7 3)Glcβ1- SG42 —Glcβ1-2(Glcβ1-3)Glcβ1- Glcβ1-3Glcβ1-2(Glcβ1- 7 3)Glcβ1- SG43 —Glcβ1-2(Glcb1-2Glcβ1- Glcβ1-2(Glcβ1-3)Glcβ1- 7 3)Glcβ1- SG44 —Glcβ1-2(Glcβ1-3)Glcβ1- Glcβ1-2(Glcb1-2Glcβ1- 7 3)Glcβ1- SG45 —Glcβ1-3Glcβ1-2(Glcβ1- Glcβ1-3Glcβ1-2(Glcβ1- 8 3)Glcβ1- 3)Glcβ1- SG46 —Glcβ1-2(Glcb1-2Glcβ1- Glcβ1-2(Glcb1-2Glcβ1- 8 3)Glcβ1- 3)Glcβ1- SG47 —Glcβ1-3Glcβ1-2(Glcβ1- Glcβ1-2(Glcb1-2Glcβ1- 8 3)Glcβ1- 3)Glcβ1- SG48 —Glcβ1-2(Glcb1-2Glcβ1- Glcβ1-3Glcβ1-2(Glcβ1- 8 3)Glcβ1- 3)Glcβ1-

TABLE 11 Fold-change in activity over parental enzyme for point mutantsof SrUGT85C2 (C13-O-glycosylating activity). Fold Fold increase increaseWT Pos. Mutation WT Pos. Mutation WT Pos. Mutation WT Pos. Mutationsteviolmonoside rubusoside D 2 G 1.1 A 3 S 1.1 V 13 A 1.0 V 13 A L 40 F1.1 V 13 I L 40 H 0.9 I 14 V 1.4 F 15 C 1.1 F 18 Y 1.2 S 22 G 0.6 S 22 GA 27 P 0.7 K 25 N 0.8 A 26 P 0.5 Q 32 K 1.0 K 37 R 1.0 L 39 F 1.1 Q 40 H1.1 Q 40 R 1.0 D 47 E 1.3 F 48 Y 1.4 I 49 N 1.3 N 51 K 1.4 Q 52 R 1.1 F53 L 1.4 E 55 K 1.0 S 57 R 1.0 H 60 N 1.4 C 61 A 1.2 A 65 L 1.1 G 67 D0.8 V 77 L 0.9 S 78 P 1.0 H 79 P 0.9 P 81 D 1.0 A 83 D 1.1 S 84 A 0.9 I85 T 1.1 P 86 Q 1.2 I 87 D 0.9 R 88 I 0.9 E 89 P 1.0 L 92 C 0.9 R 93 E1.1 I 95 T 1.0 E 96 R 0.9 T 97 K 1.0 F 99 C 1.1 F 99 L F 127 W 1.1 D 101A 1.1 R 102 P 1.2 I 104 E 1.1 I 104 R 0.7 I 104 R K 134 E 1.0 V 107 L0.9 T 108 A 1.0 P 111 N 0.8 P 114 V 1.0 0.7 I 118 V 1.0 0.7 L 123 M 1.01.0 I 128 L 1.0 1.1 K 132 E 1.0 1.3 K 133 E 1.0 0.8 V 138 E 0.7 0.3 V138 R 0.6 0.2 M 139 V 1.0 0.8 M 140 L 0.9 0.7 Y 141 F 1.0 0.9 A 145 S1.0 1.0 F 152 Y 0.8 0.4 Y 153 L 0.9 0.9 I 155 Y 1.1 0.9 H 156 R 1.0 1.1F 163 L 1.2 1.2 A 169 E 1.1 1.2 V 184 I 1.0 1.0 E 188 K 0.9 0.9 G 189 N1.1 1.1 F 195 L 1.0 0.9 L 197 F 0.8 0.6 D 198 I 1.0 0.5 W 199 R 1.0 0.8S 200 T 1.0 0.9 L 203 P 0.8 0.7 K 206 I 1.1 0.5 V 207 M 0.9 0.5 M 209 N1.4 0.9 A 214 E 1.0 0.5 P 215 T 1.8 0.9 Q 216 E 1.2 0.6 S 218 A 1.1 0.7V 221 A 1.1 0.7 H 223 A 1.2 0.8 H 224 I 0.6 0.3 I 225 L M 472 F 0.8 0.4F 226 L 1.0 0.7 H 227 N 0.1 0.0 S 235 D 1.2 0.8 I 236 V 1.2 0.7 I 237 L1.2 0.8 K 238 D 1.3 0.9 T 239 A 1.1 0.9 L 242 S 1.4 1.1 R 243 I 0.8 0.6Y 244 L 1.3 0.7 N 245 P 1.1 1.3 H 246 P 0.7 0.6 I 247 V 0.9 0.7 D 258 N0.9 0.7 F 285 L 0.7 0.9 Q 289 D 0.8 1.0 K 291 Q Y 326 R 1.2 1.0 K 291 QE 293 P Y 326 R 1.3 0.9 E 292 P Y 326 R 0.9 0.7 T 304 I 1.3 0.9 S 308 T1.3 0.9 D 311 Q 1.3 1.0 M 312 L 1.1 0.9 M 312 L I 331 V 1.6 1.1 G 316 A1.1 1.0 A 320 E H 350 R 1.2 0.8 A 320 E E 346 G H 350 R 1.3 0.6 N 323 G1.2 1.1 Y 325 P 1.1 1.0 I 329 V 1.1 1.0 S 332 P 1.3 1.0 N 333 D 1.3 1.2N 339 S 0.9 0.9 E 345 G 0.9 0.9 E 345 G H 350 R 1.4 0.9 L 346 F 0.9 0.7L 346 F I 351 T 0.9 0.7 H 349 E 1.5 1.0 K 352 D 0.9 0.8 F 355 L 1.1 0.8S 361 P 1.0 0.8 K 364 Q 0.8 0.9 L 375 V 0.9 1.0 L 375 V I 395 V 0.9 0.8L 375 V I 395 V L 432 A L 436 V 0.2 0.7 G 381 N 1.0 0.1 I 384 L 1.3 0.7L 387 I 0.8 0.9 L 387 I V 416 I 0.9 0.7 S 388 C 0.8 0.7 I 394 V L 432 A0.6 0.7 I 394 V L 436 V 1.2 C 395 A 1.4 C 395 A C 407 A 1.4 C 395 T C407 A 1.5 Y 398 F 0.9 S 399 F 1.0 W 400 A 0.9 0.3 L 403 Q 1.0 I 409 V0.9 0.9 E 414 G 1.3 E 414 K K 443 E 0.4 V 415 I 0.5 L 417 M 0.7 M 419 I1.0 0.8 G 420 D 1.2 K 422 D 0.6 D 426 E 1.0 K 429 E 1.0 K 429 E Q 434 R0.9 Q 433 R 0.4 G 439 K 0.9 H 441 K 1.0 1.0 K 442 E 0.9 1.1 K 446 R D450 E 1.0 1.0 D 449 E 0.8 W 450 L 1.1 E 452 K 0.9 1.0 K 453 L 1.1 R 455E 0.7 I 456 E 0.9 I 458 T 1.0 N 461 G 1.1 N 461 G S 466 Y 0.9 0.7 I 468L 0.4 M 471 L 0.7 I 475 V 1.0 T 476 L 0.5 V 477 L 0.6

TABLE 12 Fold-change in activity over parental enzyme for point mutantsof MbUGT1-2 (1-2′ glycosylating activity). Wild-type Wild-type Foldincrease Fold increase residue Position Mutation Insertion residuePosition Mutation steviolbioside RebD S 14 W 1.8 2.4 S 14 Y 1.7 2.3 V 89A G 185 A 1.1 0.6 V 365 I 1.4 1.9 E 366 P 2.0 3.0 V 395 Y F 396 T 0.10.2 G 417 T 0.0 0.0 H 420 E 1.5 1.8 M 421 F 1.2 1.7 M 421 I 0.3 0.3 M421 A 0.7 0.4 S 424 D 1.6 2.1 S 424 Y 0.8 0.4 GPS 0.9 1.5 between 425and 426 D 427 E 1.2 1.5 D 427 S 1.1 1.0 D 427 W R 428 E 1.4 1.9 R 428 H1.0 1.1 R 428 W 0.8 0.5 E 431 A E 431 Y 1.5 1.7 R 432 H 1.8 2.7 R 432 W1.7 2.7 K 463 D 1.3 1.4 K 463 E 1.4 2.0

TABLE 13 Fold-change in activity over parental enzyme for point mutantsof SrUGT76G1 (1-3′ glycosylating activity). Fold Fold increase increaseWT Pos. Mut. Insert. Delet. WT Pos. Mut. Delet. WT Pos. Mut. Delet. WTPos. Mut. RebA RebM F 22 V 0.3 0.1 S 77 A 1.4 1.5 N 78 A 1.1 1.2 T 81 A1.0 1.3 H 82 A 1.4 0.7 G 87 V 0.0 0.1 G87-P91 0.1 1.0 I 90 V 1.1 0.4 P91 A 0.6 0.5 I 93 V 1.0 0.3 N 94 G 1.3 0.5 L 126 G 0.5 0.4 W 127 F 0.90.2 M 145 V 0.5 0.4 S 147 G 1.2 0.4 N 151 A 0.7 0.4 H 155 A 0.7 0.3 Y155 Y 1.4 0.2 S 192 A 1.0 1.1 Y 194 G 0.8 0.1 G 2.4 1.1 between 194 and195 W 197 P 1.7 0.7 I 199 A 0.9 0.1 L 200 A T 284 A 8.2 8.6 L 200 A L379 G T 284 A S 192 A 7.0 9.0 L 200 A L 379 G T 284 A 9.4 9.2 L 200 A L379 G G87- 0.5 P91 L 200 A L 379 G 3.1 1.8 L 200 A 8.0 17.0 L 200 G 6.73.9 L 200 V 1.1 0.1 L 200 A G87- 1.6 1.6 P91 E 202 D 1.2 0.1 I 203 A 1.20.2 L 204 A 0.6 0.2 G 205 R K 206 A K 209 E 0.8 0.1 G 205 A 1.4 0.4 M207 A 0.8 0.6 T 284 A 1.0 1.5 T 284 V 1.6 0.3 L 379 G T 284 A 1.1 1.5 L379 G 0.6 2.0 L 379 A 1.9 1.4 L 397 V 2.1 0.9

The invention claimed is:
 1. A method for making Rebaudioside M (RebM)or Rebaudioside D (RebD), comprising: providing a host cell producingRebM or RebD from steviol through a plurality of uridine diphosphatedependent glycosyltransferase enzymes (UGT), the UGT enzymes comprisingone or more of: (a) a modified UGT enzyme having an increase in 1-2′glycosylating activity at C19 of Rebaudioside A (RebA) as compared toits parent UGT enzyme, without substantial loss of 1-2′ glycosylatingactivity at C13 of steviolmonoside as compared to its parent UGT enzyme,and (b) a modified UGT enzyme having an increase in 1-3′ glycosylatingactivity at C19 of Rebaudioside D (RebD) as compared to its parent UGTenzyme, without substantial loss of 1-3′ glycosylating activity at C13of stevioside as compared to its parent UGT enzyme; and culturing thehost cell under conditions for producing the RebM or RebD; wherein theUGT enzyme having an increase in 1-2′ glycosylating activity at C19 ofRebA is a circular permutant of its parent UGT enzyme and the UGT enzymehaving an increase in 1-3′ glycosylating activity at C19 of RebD is acircular permutant of its parent UGT enzyme.
 2. The method of claim 1,wherein: (a) the 1-2′ glycosylating activity at C19 of Rebaudioside A(RebA) is equal to or better than the 1-2′ glycosylating activity at C13of steviolmonoside: and/or (b) the 1-3′ glycosylating activity at C19 ofRebaudioside D (RebD) is equal to or better than the 1-3′ glycosylatingactivity at C13 of stevioside.
 3. The method of claim 1, wherein thecell expresses only one UGT enzyme having 1-2′ glycosylating activity atC13 of steviolmonoside, and/or expresses only one UGT enzyme having 1-3′glycosylating activity at C13 of stevioside.
 4. The method of claim 1,wherein the cell expresses both the UGT enzyme having an increase in1-2′ glycosylating activity at C19 of RebA that is a circular permutantof its parent UGT enzyme and the UGT enzyme having an increase in 1-3′glycosylating activity at C19 of RebD that is a circular permutant ofits parent UGT enzyme.
 5. The method of claim 1, wherein the UGT enzymehaving 1-2′ glycosylating activity is OsUGT1-2 or SrUGT91D2, orSrUGT91D1, or derivative thereof having increased glycosylating activityat C19 of RebA as compared to its parent UGT enzyme.
 6. The method ofclaim 5, wherein the UGT enzyme having 1-2′ glycosylating activity is acircular permutant of OsUGT1-2 comprising one or more amino acidsubstitutions, deletions, and/or insertions that increase 1-2′glycosylating activity at C19 of RebA as compared to its parent UGTenzyme.
 7. The method of claim 6, wherein the UGT enzyme having 1-2′glycosylating activity is a circular permutant of OsUGT1-2 having a cutsite corresponding to a position within 190 to 210 of SEQ ID NO: 7, andhaving a linker sequence between the amino acids that correspond to theN-terminal and C-terminal residues of SEQ ID NO: 7, and wherein thelinker optionally has a length of from 2 to 25 amino acids.
 8. Themethod of claim 5, wherein the UGT enzyme having 1-2′ glycosylatingactivity is SrUGT91D2, and which is a circular permutant comprising oneor more amino acid substitutions, insertions, and/or deletions thatincrease glycosylating activity of C19 of RebA as compared to its parentUGT enzyme.
 9. The method of claim 1, wherein the UGT enzyme having 1-2′glycosylating activity is MbUGT1,2-2 (SEQ ID NO: 45) or derivativethereof.
 10. The method of claim 1, wherein the UGT enzyme having 1-3′glycosylating activity is a derivative of SrUGT76G1, which is a circularpermutant comprising one or more amino acid substitutions, insertions,and/or deletions increasing glycosylation activity of C19 of RebD ascompared to its parent UGT enzyme.
 11. The method of claim 1, whereinthe UGT enzyme having 1-3′ glycosylating activity is a derivative ofSrUGT76G1 that includes an amino acid substitution at one or more ofpositions 200, 284, and/or 379, wherein amino acid positions arenumbered according to SEQ ID NO:3.
 12. The method of claim 1, whereinthe host cell further comprises a UGT enzyme that converts steviol tosteviolmonoside.
 13. The method of claim 12, wherein the UGT enzyme thatconverts steviol to steviolmonoside is SrUGT85C2, or derivative thereof.14. The method of claim 1, wherein the host cell further comprises a UGTenzyme that converts steviolbioside to stevioside.
 15. The method ofclaim 14, wherein the UGT enzyme that converts steviolbioside tostevioside is SrUGT74G1, or derivative thereof.
 16. The method of claim1, wherein the host cell produces steviol substrate through an enzymaticpathway comprising a kaurene synthase (KS), kaurene oxidase (KO), and akaurenoic acid hydroxylase (KAH), the host cell further comprising acytochrome P450 reductase (CPR) for regenerating one or more of the KOand KAH enzymes.
 17. The method of claim 1, wherein the host cellexpresses a geranylgeranyl pyrophosphate synthase (GGPPS), which isoptionally of Taxus canadensis, Abies grandis, Aspergillus nidulans,Stevia rebaudiana, Gibberella fujikuroi, Marine bacterium 443,Paracoccus haeundaensis, Chlorobium tepidum, Mus musculus, Thalassiosirapseudonana, Streptomyces melanosporofaciens, Streptomyces clavuligerus,Sulfulubus acidocaldarius, Synechococcus sp., Synechocystis sp.,Arabidopsis thaliana, Thermotogo maritime, Corynebacterium glutamicum,Therms thermophillus, Pyrobaculum calidifontis, or derivatives thereof.18. The method of claim 1, wherein the host cell expresses a pathwayproducing iso-pentenyl pyrophosphate (IPP) and dimethylallylpyrophosphate (DMAPP).
 19. The method of claim 18, wherein the host cellis prokaryotic or eukaryotic.
 20. The method of claim 19, wherein thehost cell is E. coli, Bacillus subtillus, or Pseudomonas putida.
 21. Themethod of claim 19, wherein the host cell is a species of Saccharomyces,Pichia, or Yarrowia, including Saccharomyces cerevisiae, Pichiapastoris, and Yarrowia lipolytica.
 22. The method of claim 1, whereinthe host cell comprises the following heterologously expressed genes:Taxus canadensis GGPPS or derivative thereof, Phaeosphaeria sp. PsCK (acopalyl diphosphate-kaurene synthase) or derivative thereof, Steviarebaudiana KO or derivative thereof, Arabidopsis thaliana KAH orderivative thereof, Stevia rebaudiana CPR or derivative thereof, Steviarebaudiana UGT85C2 or derivative thereof, Stevia rebaudiana UGT74G I orderivative thereof or MbUGTC 19 or derivative thereof, Stevia rebaudianaUGT76G I or derivative thereof or MbUGT1-3 or derivative thereof, andStevia rebaudiana UGT91 D2 or OsUGT1-2 or MbUGT1-2 or derivativethereof.
 23. The method of claim 1, wherein the host cell comprises thefollowing heterologously expressed genes: Corynebacterium glutmnncunGGPS or derivative thereof, Erwina tracheiphila copalyl diphosphatesynthase (CPPS) or derivative thereof, Erwina tracheiphila KS orderivative thereof, Stevia rebaudiana KO or derivative thereof,Arabidopsis thaliana KAH or derivative thereof, Stevia rebaudiana CPR orderivative thereof, Stevia rebaudiana UGT85C2 or derivative thereof,Stevia rebaudiana UGT74G1 or derivative thereof or MbUGTC19 orderivative thereof or MbUGTC19-2 or derivative thereof, Steviarebaudiana UGT76G1 or derivative thereof or MbUGT1-3 or derivativethereof, and Stevia rebaudiana UGT91 D2 or OsUGT1-2 or MbUGT1,2-2 orderivative thereof.
 24. The method of claim 1, further comprisingrecovering the RebM or RebD from culture media.